Methods for determining the likelihood of survival and for predicting likelihood of metastasis in cancer patients

ABSTRACT

The present invention relates generally to methods of accurately quantifying HER2 and/or p95 expression in subjects with a HER2 positive cancer and indicating the risk of brain relapse in such patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 13/476,735, filed May 21, 2012, which claims thebenefit of and priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application Ser. No. 61/488,028, filed May 19, 2011. Theseapplications are each incorporated herein by reference in theirentireties.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 14/737,742, filed Jun. 12, 2015, which is acontinuation of U.S. patent application Ser. No. 13/911,329, filed Jun.6, 2013, and issued as U.S. Pat. No. 9,081,019 on Jul. 14, 2015, whichis a divisional of U.S. patent application Ser. No. 12/629,037, filedDec. 1, 2009, and issued as U.S. Pat. No. 8,470,542 on Jun. 25, 2013,which claims the benefit of and priority under 35 U.S.C. § 119(e) toU.S. Provisional Application No. 61/118,975, filed Dec. 1, 2008, U.S.Provisional Application No. 61/182,282, filed May 29, 2009, and U.S.Provisional Application No. 61/187,960, filed Jun. 17, 2009. Theseapplications are each incorporated herein by reference in theirentireties.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 15/681,895, filed Aug. 21, 2017, which is acontinuation of U.S. patent application Ser. No. 14/802,170, filed Jul.17, 2015, and issued as U.S. Pat. No. 9,766,242 on Sep. 19, 2017, whichis a continuation of U.S. patent application Ser. No. 13/670,508, filedNov. 7, 2012, and issued as U.S. Pat. No. 9,110,066 on Aug. 18, 2015,which is a divisional application of U.S. patent application Ser. No.12/688,798, filed Jan. 15, 2010, and issued as U.S. Pat. No. 8,349,574on Jan. 8, 2013, which claims the benefit of and priority under 35U.S.C. § 119(e) to U.S. Provisional Application No. 61/176,630, filedMay 8, 2009, U.S. Provisional Application No. 61/187,962, filed Jun. 17,2009, and U.S. Provisional Application No. 61/145,029, filed Jan. 15,2009. These applications are each incorporated herein by reference intheir entireties.

FIELD

The present invention relates generally to methods of accuratelyquantifying total HER2 or p95 expression in patients with a HER2positive cancer, such as advanced breast cancer, and correlating HER2 orp95 expression with the risk of brain relapse in such patients.

BACKGROUND

Brain metastases accompanying breast cancer are associated withparticularly poor prognosis. Brain metastases seriously affect qualityof life and are relatively resistant to systemic therapies. Breastcancer is the second most common cause of brain metastases. Though thebiological basis is not yet fully understood, patients withHER2-positive breast cancer are at a particularly high risk of brainmetastases. However, currently there are no clinical or biologicalfeatures that have been shown to consistently associate with apropensity to develop brain relapse in HER2-positive advanced breastcancer patients. Similarly, no robust molecular marker to predict brainrelapse has been developed.

Current methodologies for determining of HER2 status includeimmunohistochemistry (IHC) to detect HER2 protein overexpression,fluorescence in situ hybridization (FISH), or chromogenic in situhybridization to detect amplification of the HER2 gene. However,considerable controversy still exists regarding the accuracy,reliability, and inter-observer variability of these assay methods. Forexample, the assessment of HER2 expression by IHC is inherentlysubjective and semi-quantitative (scored as 0, 1+, 2+, 3+). The FISHtest, where HER2 gene copy number is counted, is more quantitativeanalytically, but multiple clinical studies have failed to demonstrate arelationship between HER2 gene copy number and response to clinicaltreatment. Using currently available techniques, it is estimated thatapproximately 20% of HER2 testing may be inaccurate.

Currently, the standard component of systemic therapy in HER2-positivebreast cancer patients is trastuzumab, a monoclonal antibody against theextracellular domain of the HER2 receptor. However, due to a highmolecular weight (approx. 145,000 Da), and physical and chemicalproperties, trastuzumab does not cross the blood-brain barrier and isineffective in preventing and treating brain metastases. In addition, asubgroup of HER2-overexpressing tumors also have p95 HER2 (p95), anN-terminal truncated version of HER2 that has shed the ectodomain. Astrastuzumab binds to the ectodomain of HER2, it cannot bind the p95truncated HER2 protein, so trastuzumab is ineffective in patients withhigh levels of p95.

Because cancer is such a complex disease, many new targeted therapies,while extremely effective in some individual patients, have limitedeffectiveness in the general patient population for a particular cancer.Without appropriate knowledge about the patient, healthcare providersmay be unable to select an effective targeted therapy for the patient.For example, some patients with advanced breast cancer may be at anincreased risk of brain metastasis, but the treatment that they arereceiving may be inappropriate for preventing or treating brainmetastasis.

Therefore, what is needed is a method for accurately quantifying totalHER2 or p95 expression and a method to correlate HER2 or p95 level withthe risk of brain relapse in patients with advanced breast cancer. Whatare also needed are methods for identifying therapeutic treatmentstrategies that are specifically tailored to the subpopulation ofHER2-positive advanced breast cancer patients. Methods for selecting acourse of treatment for an individual with a HER2-positive cancer andfor monitoring the progress of a course of treatment are also needed.

SUMMARY

The present invention relates generally to methods of accuratelyquantifying total HER2 or p95 expression in patients with advancedbreast cancer and correlating HER2 or p95 expression with the risk ofbrain relapse and time to brain metastasis (TTBM) in such patients.Embodiments of the invention described herein include methods thatutilize VeraTag® technology to accurately quantify total HER2 and p95protein expression in tumor samples and correlate HER2 level and therisk of brain relapse in HER2-positive advanced breast cancer patients,including patients that received treatment, such as trastuzumab.

Embodiments of the invention include a novel assay that preciselyquantifies total HER2 expression (H2T), p95 and HER2 homodimers (H2D) inbiological samples.

BRIEF DESCRIPTION OF THE FIGURES

Non-limiting embodiments of the methods and systems of the invention areexemplified in the following figures.

FIG. 1 shows two exemplary assay formats based on VeraTag® technologyaccording to embodiments of the invention. Panel A depicts a standardHerMark® assay format, which is a proximity-based assay where cleavageof the VTag® reporter molecule occurs via singlet oxygen. The symbol ofthe scissors represents the cleavage moiety attached to the cleavageagent, while “hv” represent light energy. In Panel B, cleavage of theVTag® reporter molecule is effected by a reducing agent (e.g., DTT). Inboth assay formats, released VTag® reporter molecules are separated andidentified by capillary electrophoresis. The x-axis shows the time atwhich the cleaved VTag® reporter molecule eluted from the capillary, andthe fluorescence intensity is shown on the y-axis. Peaks denote theelution of different VTag® reporter molecules.

FIG. 2 shows exemplary assay formats based on VeraTag® technologyaccording to embodiments of the invention. Panel A depicts a standardHerMARK® assay format, which enables quantitation of the total amount oftarget protein (e.g., HER2) using two binding agents (e.g., antibodies)specific for the target protein. Panel B depicts an assay format thatenables identification of homodimers using a single antibody that hasbeen differentially conjugated to either a VTag® reporter molecule or acleaving agent. Both assay formats are proximity-based assays wherecleavage of the VTag® reporter molecule occurs via photo-induction ofthe cleaving agent (i.e., singlet oxygen) by light. “S” represents astreptavidn molecule attached via a biotin molecule to one of theantibodies. Panel C shows an exemplary VTag® reporter molecule (Pro11)attached to an antibody (e.g., Ab8) via a cleavable linker and inreleased form after cleavage.

FIG. 3 is a graph illustrating the relationship between quantitativeHER2 protein as measured by H2T (RF/mm² tumor) in a HERmark® assay andHER2 gene copy number per centromere 17 as measured by HER2 FISH.

FIG. 4 shows Kaplan Meier plots illustrating time to brain metastases inmonths based on different protein marker categories according toembodiments of the invention. Panel A shows data based on quantitativeHER2 levels, wherein subjects with a H2T level above (grey line) andbelow (black line) the median are compared. Panel B shows data based onquantitative HER2 levels in subjects with levels of HER2 above (blackline) and below (grey line) the optimal cutoff of H2T. Panel C showsdata based on HER2 FISH analysis, where subjects with levels above(black line) vs. below (grey line) the median are compared. Panel Dshows data based on quantitative p95 levels, wherein subjects with a p95level above (grey line) and below (black line) an optimal p95 cutoff arecompared. Panel E shows data based on quantitative p95 levels within thelow HER2 group identified in Panel B to illustrate the independence ofH2T and p95 as significant biomarkers.

FIG. 5 illustrates the effect of quantitative HER2 levels in view oftumor grade on time to brain metastases according to embodiments of theinvention. Panel A shows a Forest plot illustrating the hazard ratiodetermined based on quantitative HER2 levels and tumor grade. Panel B isa Kaplan-Meier plot illustrating the time to brain metastases in monthswhen HER2 levels in different tumor grades are compared.

FIG. 6 illustrates the effect of quantitative HER2 levels in view oftumor grade on time to brain metastases according to embodiments of theinvention. Panel A is a Forest plot illustrating the hazard ratio basedon quantitative continuous HER2 levels (no cutoff) and tumor grade.Panel B is a Forest plot illustrating the hazard ratio based onquantitative continuous HER2 levels and tumor grade within the subset ofHER2 FISH positive subjects.

FIG. 7 is a diagram showing the dominant site of metastasis asdetermined by a physician at the beginning of the study and the H2Tlevel for each of the dominant metastatic sites.

DETAILED DESCRIPTION Definitions and Abbreviations

As used herein, the terms “embodiment” and “aspect” are usedinterchangeably.

The term “about,” as used herein, unless otherwise indicated, refers toa value that is not more than 10% above or below the value beingmodified by the term. For example, the term “about 5 μg/kg” means arange of from 4.5 μg/kg to 5.5 μg/kg. As another example, “about 1 hour”means a range of from 48 minutes to 72 minutes.

“Antibody” means an immunoglobulin that specifically binds to, and isthereby defined as complementary with, a particular spatial and polarorganization of another molecule. The antibody can be monoclonal,polyclonal, or recombinant and can be prepared by techniques that arewell known in the art such as immunization of a host and collection ofsera (polyclonal) or by preparing continuous hybrid cell lines andcollecting the secreted protein (monoclonal), or by cloning andexpressing nucleotide sequences or mutagenized versions thereof codingat least for the amino acid sequences required for specific binding ofnatural antibodies. Antibodies may include a complete immunoglobulin orfragment thereof, which immunoglobulins include the various classes andisotypes, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, IgM, etc.Fragments thereof may include Fab, Fv, and F(ab′)₂, Fab′, and the like.Antibodies may also be single-chain antibodies or an antigen-bindingfragment thereof, chimeric antibodies, humanized antibodies or any otherantibody derivative known to one of skill in the art that retainsbinding activity that is specific for a particular binding site. Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity for a particular binding site is maintained. Guidance in theproduction and selection of antibodies and antibody derivatives for usein immunoassays, including such assays employing releasable moleculartag (as described below) can be found in readily available texts andmanuals, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York; Howard and Bethell, 2001,Basic Methods in Antibody Production and Characterization, CRC Press;Wild, ed., 1994, The Immunoassay Handbook, Stockton Press, New York.

“Antibody binding composition” means a molecule or a complex ofmolecules that comprises one or more antibodies, or antigen-bindingfragments that bind to a molecule, and derives its binding specificityfrom such antibody or antibody-binding fragment. Antibody bindingcompositions include, but are not limited to, (i) antibody pairs inwhich a first antibody binds specifically to a target molecule and asecond antibody binds specifically to a constant region of the firstantibody; a biotinylated antibody that binds specifically to a targetmolecule and a streptavidin protein, which protein is derivatized withmoieties such as molecular tags or photosensitizers or the like, via abiotin moiety; (ii) antibodies specific for a target molecule andconjugated to a polymer, such as dextran, which, in turn, is derivatizedwith moieties such as molecular tags or photosensitizers, eitherdirectly by covalent bonds or indirectly via streptavidin-biotinlinkages; (iii) antibodies specific for a target molecule and conjugatedto a bead, or microbead, or other solid phase support, which, in turn,is derivatized either directly or indirectly with moieties such asmolecular tags or photosensitizers, or polymers containing the latter.

“Antigenic determinant,” or “epitope” means a site on the surface of amolecule, usually a protein, to which a single antibody molecule binds.Generally, a protein has several or many different antigenicdeterminants and reacts with antibodies of different specificities. Apreferred antigenic determinant is a phosphorylation site of a protein.

“Binding compound” shall refer to an antibody binding composition, anantibody, a peptide, a peptide or non-peptide ligand for a cell surfacereceptor, a protein, an oligonucleotide, an oligonucleotide analog, suchas a peptide nucleic acid, a lectin, or any other molecular entity thatis capable of specifically binding to a target protein or molecule orstable complex formation with an analyte of interest, such as a complexof proteins. In one aspect, a binding compound, which can be representedby the formula below, comprises one or more molecular tags attached to abinding moiety.

As used herein, the “blood-brain barrier” refers to a separation ofcirculating blood from the brain extracellular fluid (BECF) in thecentral nervous system (CNS). The blood brain barrier is both a physicalbarrier and a system of cellular transport mechanisms. Endothelial cellsrestrict the diffusion of microscopic objects (e.g., bacteria) and largeor hydrophilic molecules into the cerebrospinal fluid, while allowingthe diffusion of small hydrophobic molecules.

“Binding moiety” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of specifically bindingto an analyte. Binding moieties include, but are not limited to,antibodies, antibody binding compositions, antibody fragments, peptides,and proteins. Preferably, binding moieties are antibodies or antibodybinding compositions.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in an organism, such as a mammal, that is typicallycharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma andleukemia. More particular examples of such cancers include squamous cellcancer, lung cancer, e.g., small-cell lung cancer or non-small cell lungcancer; gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial carcinoma,salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, and various types of head and neckcancer.

“Chemotherapeutic agent” means a chemical substance, primarily acytotoxic or cytostatic agent, that is used to treat a condition,particularly cancer. Chemotherapeutic agents shall include suchcompounds as paclitaxel, as set forth herein.

A “cleavable linkage,” as used herein, refers to a chemical linkinggroup that may be cleaved under conditions that do not degrade thestructure or affect detection characteristics of a molecular tagconnected to a binding moiety with the cleavable linkage.

A “cleavage-inducing moiety,” or “cleaving agent,” as used herein, is agroup that produces an active species that is capable of cleaving acleavable linkage, for example by oxidation. Preferably, the activespecies is a chemical species that exhibits short-lived activity so thatits cleavage-inducing effects are only in the proximity of the site ofits generation.

A “cleaving probe,” as used herein, refers to a reagent that comprises acleavage-inducing moiety as defined herein and an antibody bindingcomposition, an antibody, an antibody fragment, a peptide or non-peptideligand for a cell surface receptor, or a protein, such as biotin orstreptavidin, an oligonucleotide, a lectin or any other molecular entitythat is capable of specifically binding to a target protein or moleculeor stable complex formation with an analyte of interest (e.g., proteinor protein complex).

As used herein, “cutoff” refers to a mathematically determined pointbased on the measurement of the amount of HER2 or p95 protein inbiological samples that can divide samples in a subject population intotwo distinct patient subgroups, such as, e.g., a median or an optimal orpredetermined cutoff. The amount can be measured by any method known inthe art such as Fluorescence resonance energy transfer (FRET),Biolumenescent resonance energy transfer (BRET), proximity ligationassay (PLA), dimer-specific antibodies, or VeraTag® assay, or any othermethod that is well known to one skilled in the art.

“VeraTag® assay” and “VERATAG® assay” are used interchangeably and referto single and multiplexed and multi-label assays, materials, methods andtechniques for performing and utilizing such assays, including but notlimited to reagents, analytical procedures and software related to thoseassays. Such assays are disclosed in this application as well as in U.S.Pat. No. 7,105,308 and in U.S. Patent Publication Nos. 2009/0191559,2010/0143927, 2010/0210034, and 2010/0233732, which is incorporated byreference herein including any drawings.

As used herein, “VeraTag® reporter molecule” or “vTag,” or “vTag®,” areused to refer to a molecular tag that is attached to an antibody used ina VeraTag® assay.

As used herein, “greater than or equal to” (i.e., ≥ or >=) can incertain alternative embodiments mean greater than (>). Also, as usedherein, “less than or equal to” (i.e., ≤ or <=) can in certainalternative embodiments mean less than (<).

“FFPE” shall refer to a group of cells or quantity of tissue that arefixed, particularly conventional formalin-fixed paraffin-embeddedsamples. Such samples are typically, without limitation, used in anassay for receptor complexes in the form of thin sections, e.g. 3-10 μmthick, of fixed tissue mounted on a microscope slide or equivalentsurface. Such samples also typically undergo a conventional re-hydrationprocedure, and optionally, an antigen retrieval procedure as a part of,or preliminary to, assay measurements.

“Hazard ratio” or “HR”, as used herein, refers to a specific type ofrelative risk that is calculated using a statistical technique known asSurvival Analysis (e.g., analysis of one or more subject groups thathave different times to some event or outcome of interest). Survivalanalysis keeps track of how many subjects have not experienced the eventat a given time or during a given time interval. The data is thenplotted over the entire time of the study, and the results are graphedas a decreasing curve. “Hazard ratio” is the ratio between the predictedhazard of one group versus another group. The hazard ratio can then becompared to an independent measure (e.g., the amount of one or morebiomarkers present in the sample; tumor grade). A hazard ratio greaterthan one indicates that event of interest is happening faster for afirst group than for a second group. A hazard ratio less than oneindicates that the event of interest is happening slower for the firstgroup than for the second group. If the HR is indistinguishable fromone, there is no statistical difference between the risk associated withthe two variables. Note that these ratios are comparisons between thetwo groups and give no indication of how long it takes for the averagesubject in either group. The hazard ratio refers to the fold increasedor decreased risk of the event of interest in the first group comparedto the second group.

“Her-2,” “ErbB2,” “c-Erb-B2,” “HER2,” “Her2,” and “neu” are usedinterchangeably herein and refer to native HER2, and allelic variantsthereof, as described, for example, in Semba et al., 1985, P.N.A.S. USA82:6497-650 and Yamamoto et al., 1986, Nature 319:230-234 and Genebankaccession number X03363. Unless indicated otherwise, the terms “HER2,”“ErbB2,” “c-Erb-B2,” “HER2,” and “Her2” when used herein refer to thehuman protein. The gene encoding Her2 is referred to herein as “erbB2.”As used herein, H2T shall refer to total HER2 expression as determined,for example without limitation, by VeraTag® assay.

“HER2-acting agent,” as used herein, refers to a compound that caninhibit a biological activity of HER2 or a HER2 expressing cell or aHER2 positive cancer cell. Such biological activities include, but arenot limited to, dimerization, autophosphorylation, phosphorylation ofanother receptor, signal transduction, and the like. Biologicalactivities can include, without limitation, cell survival and cellproliferation, and inhibition of such activities by a HER2 acting agentcould be direct or indirect cell killing (e.g., ADCC), disruption ofprotein complexes or complex formation, modulation of proteintrafficking, enzyme inhibition or down regulation of HER2. Biologicalactivities can also include patient response as set forth in thisapplication. Exemplary HER2-acting agents include, but are not limitedto, the antibodies pertuzumab, ertumaxomab, and trastuzumab and smallmolecules such as 17-AAG, IPI-504, neratinib, AZD8931, ARRY-380, PF299,afatinib, pelitinib, S-222611, AEE-788 and lapatinib. Antibodies andrelated molecules are generally too large to pass through theblood-brain barrier.

“HER2 homodimer” in reference to cell surface HER2 membrane receptorsmeans a complex of two or more membrane-bound HER2 proteins. Dimersusually consist of two receptors in contact with one another. Dimers maybe created in a cell surface membrane by passive processes, such as Vander Waal interactions, and the like, or dimers may be created by activeprocesses, such as by ligand-induced dimerization, covalent linkages,interaction with intracellular components or the like. See, e.g.,Schlessinger, 2000, Cell 103:211-225. As used herein, the term “dimer”is understood to refer to “cell surface membrane receptor dimer,” unlessunderstood otherwise from the context. As used herein, “H2D” shall referto quantified dimer as determined, for example without limitation, byVeraTag® assay.

A “HER2 positive” cancer, cancer cell, subject or patient, as usedherein, refers to a cancer, cancer cell, subject, or patient exhibitinga score of at least 2 when using a HercepTest® (DakoCytomationCalifornia Inc., Carpenteria, Calif.) or a cancer, cancer cell, subject,or patient that has been identified as such by FISH, having a centromere17 corrected HER2 gene copy number greater than 2 (HER2 FISH/CEP17>2;FISH positive). In certain embodiments, the HER2 positive cell exhibitsa score of at least 2+ or 3+ using the HercepTest® Immunohistochemistryassay.

“High” refers to a measure that is greater than normal, greater than astandard (e.g., a predetermined measure or a subgroup measure), or thatis relatively greater than another subgroup measure. For example, highHER2 refers to a measure of HER2 that is greater than a normal HER2measure. A normal HER2 measure may be determined according to any methodavailable to one skilled in the art. High HER2 may also refer to ameasure that is equal to or greater than a predetermined measure, suchas a predetermined cutoff High HER2 also may refer to a measure of HER2wherein a high HER2 subgroup has relatively greater levels of HER2 thananother subgroup. For example, without limitation, according to thepresent invention, two distinct patient subgroups can be created bydividing samples around a mathematically determined point, such as,without limitation, a median, thus creating a subgroup whose measure ishigh (i.e., higher than the median) and another subgroup whose measureis low. HER2 can be measured by any method known to one skilled in theart such as, for example, without limitation, using a VeraTag® assay, orusing any standard immunohistochemical (IHC) method such as HercepTest®.As another example, high HER2 can refer to a measure of HER2 that isgreater than a normal measure of HER2 in a particular set of samples orpatients that are HER2 positive. A normal HER2 measure may be determinedaccording to any method available to one skilled in the art. As anotherexample, high levels of HER2 may also refer to a measure that is greaterthan a predetermined measure, such as a predetermined cutoff High HER2also may refer to a measure of HER2 wherein a high HER2 homodimersubgroup has a relatively higher level of HER2 homodimers than anothersubgroup. In some circumstances, “high” refers to an amount that isgreater than the median measurement in a reference group.

“Likely to,” as used herein, refers to an increased probability that anitem, object, thing or event will occur.

“Long,” as used herein, refers to a time measure that is greater thannormal, greater than a standard such as a predetermined measure, or asubgroup measure that is relatively longer than another subgroupmeasure. For example, with respect to a patient's longevity, a long timeprogression refers to time progression that is longer than a normal timeprogression or longer than time to progression as compared to anothergroup. Whether a time progression is long or not may be determinedaccording to any method available to one skilled in the art. Long couldinclude, for example, no progression. In some circumstances, “long”refers to a time that is greater than the median time course requiredfor a significant event to occur in a disease.

“Low” is a term that refers to a measure that is less than normal, lessthan a standard (e.g., a predetermined measure or a subgroup measure),or that is relatively less than another subgroup measure. For example,low HER2 means a measure of HER2 that is less than a normal HER2 measurein a particular set of samples of patients that is HER2 positive. Anormal HER2 measure may be determined according to any method availableto one skilled in the art. Low HER2 may also mean a method that is lessthan a predetermined measure, such as a predetermined cutoff. Low HER2may also mean a measure wherein a low HER2 subgroup is relatively lowerthan another subgroup. For example, without limitation, according to thepresent specification, two distinct patient subgroups can be created bydividing samples around a mathematically determined point, such as,without limitation, a median, thus creating a group whose measure is low(i.e., less than the median) with respect to another group whose measureis high. As another example, low HER2 means a measure of HER2 homodimersthat is less than a normal measure of HER2 in a particular set ofsamples or patients that is HER2 positive. Low HER2 may also mean ameasure that is less than a predetermined measure, such as apredetermined cutoff. Low HER2 may also mean a measure wherein a lowHER2 subgroup is relatively less than another subgroup.

A “molecular tag,” as used herein, refers to a molecule that can bedistinguished from other molecules based on one or more physical,chemical, or optical differences among the molecules being separated,including but not limited to, electrophoretic mobility, molecularweight, shape, solubility, pKa, hydrophobicity, charge, charge/massratio, polarity, or the like. In one aspect, molecular tags in aplurality or set differ in electrophoretic mobility and opticaldetection characteristics and can be separated by electrophoresis. Inanother aspect, molecular tags in a plurality or set may differ inmolecular weight, shape, solubility, pKa, hydrophobicity, charge, orpolarity and can be separated by normal phase or reverse phase HPLC, ionexchange HPLC, capillary electrochromatography, mass spectroscopy, gasphase chromatography or like technique. As described herein, a VeraTag®reporter molecule is a type of molecular tag

“Optimal cutoff” or “optimized cutoff”, as used herein, refers to thevalue of a predetermined measure in subjects exhibiting certainattributes that allow the best discrimination between two categories ofan attribute. For example, finding a value for an optimal cutoff thatallows one to best discriminate between two categories (e.g., high H2Texpression and low H2T expression) for determining, e.g., overallsurvival (OS). Optimal cutoffs are used to separate the subjects withvalues lower than or higher than the optimal cutoff to optimize aprediction model (e.g., for example, without limitation, to maximize thespecificity of the model, maximize the sensitivity of the model,maximize the difference in outcome, or minimize the p-value from hazardratio or a difference in response).

“Overall survival” or “OS” refers to a time as measured from the startof treatment to death or censor. Censoring may come from a study end orchange in treatment. Overall survival can refer to a probability as, forexample, a probability when represented in a Kaplan-Meier plot of beingalive at a particular time, that time being the time between the startof the treatment to death or censor.

As used herein, “p95” refers to an N-terminally truncated, C-terminalportion of HER-2. “p95” has also been referred to as “truncated ErbB2receptor”, “p95^(ErbB2)”, “p95HER2”, and more generally as“NH₂-terminally truncated HER-2/neu” and “HER2 C-terminal fragments” toreflect the fact that “p95” represents a family of truncated HER2proteins similar, but not identical in size to that originallyidentified as having an apparent molecular weight of 95 kiloDaltons. p95is thought to be produced by at least two distinct mechanisms. p95 mayresult from the proteolytic cleavage of full-length HER-2. p95 may alsoresult from an alternative translational start downstream from thecanonical first methionine including but not limited to M611 and M687.

As used herein, “photosensitizer” refers to a light-adsorbing moleculethat when activated by light converts molecular oxygen into singletoxygen.

“RECIST” shall mean an acronym that stands for “Response EvaluationCriteria in Solid Tumours” and is a set of published rules that definewhen cancer patients improve (“respond”), stay the same (“stable”) orworsen (“progression”) during treatments. Response as defined by RECISTcriteria have been published, (see, e.g., Therasse, 2000, J. Natl.Cancer Inst. 92(3):205-216). RECIST criteria may include other similarpublished definitions and rule sets. One skilled in the art wouldunderstand definitions that go with RECIST criteria, as used herein,such as partial response (PR), complete response (CR), stable disease(SD) and progressive disease (PD).

“Relative fluorescence units” or “RFUs” are used interchangeably andshall refer to the time integral of a particular capillaryelectrophoresis peak using arbitrary fluorescence units in comparison toa standard. With respect to VeraTag® assay formats, the RFU isproportional to the concentration of VeraTag® reporter molecule injectedinto capillary electrophoresis with some expected variability introducedby, for example, injection and capillary differences. The readout ofVeraTag® assays are generally given in units of relative fluorescenceper mm² tumor (RF/mm²).

“Relative peak area” or “RPA” are used interchangeably and shall referto the ratio between an RFU of a particular VeraTag® reporter moleculeand an RFU of a known internal fluorescence standard of known andconstant concentration.

“Responsiveness,” to “respond” to treatment, and other forms of thisverb, as used herein, refer to the reaction of a subject to treatmentwith a HER2-acting agent. As an example, a subject responds to treatmentwith a Her2-acting agent if growth of a tumor in the subject is retardedabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In anotherexample, a subject responds to treatment with a HER2-acting agent if atumor in the subject shrinks by about 5%, 10%, 20%, 30%, 40%, 50%, ormore as determined by any appropriate measure, (e.g., by mass orvolume). In another example, a subject responds to treatment with aHer2-acting agent if the subject experiences a life expectancy extendedby about 5%, 10%, 20%, 30%, 40%, 50%, or more beyond the life expectancypredicted if no treatment is administered. In another example, a subjectresponds to treatment with a HER2-acting agent if the subject has anincreased disease-free survival, overall survival or increased time toprogression. Several methods may be used to determine if a patientresponds to a treatment including the RECIST criteria, as set forthabove.

“Sample,” “tissue sample,” “biological sample,” “patient sample,”“patient cell or tissue sample,” or “specimen” each refer to acollection of similar cells obtained from a tissue of a subject orpatient. The source of the tissue sample may be solid tissue as from afresh, frozen, and/or preserved organ or tissue sample or biopsy oraspirate; blood or any blood constituents; bodily fluids such ascerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitialfluid; or cells from any time in gestation or development of thesubject. The tissue sample may contain compounds that are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.Cells may be fixed in a conventional manner, such as in an FFPE manner.

“Short,” as used herein, refers to a time measure that is shorter thannormal, shorter than a standard such as a predetermined measure or asubgroup measure that is relatively shorter than another subgroupmeasure. For example, with respect to a patient's longevity, a shorttime progression refers to time progression that is shorter than anormal time progression. Whether a time progression is short or not maybe determined according to any method available to one skilled in theart. In some circumstances, “short” refers to a time that is less thanthe median time course required for a significant event to occur in adisease.

As used herein, “significant event” shall refer to an event in apatient's disease that is important as determined by one skilled in theart. Examples of significant events include, for example, withoutlimitation, primary diagnosis, death, recurrence, the determination thata patient's disease is metastatic, relapse of a patient's disease or theprogression of a patient's disease from any one of the above notedstages to another. A significant event may be any important event usedto assess OS, time to progression (TTP) and/or using the RECIST or otherresponse criteria, as determined by one skilled in the art.

As used herein, “small molecule drug” refers to a low molecular weightorganic compound which is by definition not a polymer. The term smallmolecule, especially within the field of pharmacology, is usuallyrestricted to a molecule that also binds with high affinity to abiopolymer such as protein, nucleic acid, or polysaccharide and inaddition alters the activity or function of the biopolymer. The uppermolecular weight limit for a small molecule is approximately 1000Daltons which allows for the possibility to rapidly diffuse across cellmembranes so that they can reach intracellular sites of action.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, preferably a mammal including a non-primate (e.g., acow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse,sheep) and a primate (e.g., a monkey, such as a cynomolgous monkey,gorilla, chimpanzee and a human).

As used herein, “time course” shall refer to the amount of time betweenan initial event and one or more subsequent events. For example, withrespect to a subject' cancer, time course may relate to a patient'sdisease and may be measured by gauging significant events in the courseof the disease, wherein the first event may be diagnosis and thesubsequent event may be, e.g., but not limited to, progression to alater stage, relapse, metastasis, surgery, or death.

“Time to progression” or “TTP” refers to a time as measured from thestart of the treatment to progression or a cancer or censor. Censoringmay come from a study end or from a change in treatment. Time toprogression can also be represented as a probability as, for example, ina Kaplan-Meier plot where time to progression may represent theprobability of being progression free over a particular time, that timebeing the time between the start of the treatment to progression orcensor.

“Time to brain metastasis” or “TTBM” refers to a time as measured fromthe start of the treatment to occurrence of brain metastasis or censor.Censoring may come from a study end or from a change in treatment. Timeto brain metastasis can also be represented as a probability of brainmetastasis, as, for example, in a Kaplein-Meier plot where time to brainmetastasis may represent the probability of being brain metastasis freeover a particular time, that time being the time between the start ofthe treatment to brain metastasis or censor. TTBM is a type of TTP, asbrain metastasis may be a significant event in the time course of asubject's cancer.

“Treat,” “treatment,” and other forms of this word refer to theadministration of a Her-acting agent and/or a chemotherapeutic agentand/or other cancer treatment to impede growth of a cancer, to cause acancer to shrink by weight or volume, and/or to extend the expectedsurvival time of the subject and/or time to progression of the tumor, orthe like.

“Unlikely to” refers to a decreased probability that an event, item,object, thing or person will occur with respect to a reference.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention relates generally to methods of accuratelyquantifying total HER2 or p95 expression in patients with a HER2positive cancer, such as advanced breast cancer, and correlating HER2 orp95 expression with the risk of brain metastases in such patients. Themethods of the invention enable identification of quantitative HER2 andp95 cutoffs to enable classification of subjects into risk subgroupsbased on the amount of HER2 and/or p95 in biological samples from thesubject. The methods of the invention also enable quantitatitvemeasurement of HER2 and/or p95 characterization of a subject's risk inrelation to a subject population as a whole as well.

In certain embodiments, the invention uses clinical data in combinationwith measurements of biomarkers to predict patient outcome. The clinicaldata may include overall survival, time to brain metastasis andprogression to metastatic disease. For example, the clinical data mayinclude the date of death or the date of the patient's last follow-upappointment. Or, other aspects of clinical data (e.g., time tometastasis, time to remission, development of resistance to a particulartherapeutic agent) may be used. In certain embodiments, the clinicaldata may comprise the date of starting treatment with a particularanti-HER acting agent or a chemotherapeutic agent. In other embodiments,the invention uses clinical data in order to determine a course oftherapeutic action or treatment for a patient, particularly a patientthat is at high-risk of relapse/metastasis.

One embodiment is a method for determining the relative likelihood ofwhether a subject with a HER2− positive cancer is at risk for developingbrain metastases comprising (a) obtaining a biological sample of a tumorfrom the subject's cancer; (b) measuring the amount of at least one ofHER2 or p95 in the biological sample; (c) determining whether the amountof at least one of HER2 or p95 protein in the subject's sample is abovea HER2 cutoff or a p95 cutoff; and (d) indicating that, if the amount ofHER2 or p95 protein in the biological sample are above the HER2 cutoffor p95 cutoff, the subject is more likely to be at risk for developingbrain metastases.

Still another embodiment is a method for selecting a course of treatmentfor a subject with a HER2-2 positive cancer comprising obtaining abiological sample of a tumor from the subject's cancer, (a) obtaining abiological sample of a tumor from the subject's cancer; (b) measuringthe amount of at least one of HER2 or p95 in the biological sample; (c)determining whether the amount of at least one of HER2 or p95 protein inthe subject's sample is above a HER2 cutoff or a p95 cutoff; and (d)indicating an appropriate course of treatment for the subject based onthe amount of HER2 or p95 protein in the biological sample are above theHER2 cutoff or p95 cutoff.

In one embodiment, the invention comprises a method of identifyingsubjects with HER2 positive cancer that should be screened for brainmetastasis, comprising: (a) obtaining a biological sample of a tumorfrom the subject's cancer; (b) measuring the amount of at least one ofHER2 or p95 in the biological sample; (c) determining whether the amountof at least one of HER2 or p95 protein in the subject's sample is abovea HER2 cutoff or a p95 cutoff; and (d) indicating that the subjectshould be screened for brain metastasis if the amount of at least one ofHER2 or p95 is above the HER2 cutoff or the p95 cutoff.

In another embodiment, the invention comprises a method of identifyingsubjects with HER2 positive cancer that should receive treatment with aHER2-acting agent and a second form of cancer treatment, comprising: (a)obtaining a biological sample of a tumor from the subject's cancer; (b)measuring the amount of at least one of HER2 or p95 in the biologicalsample; (c) determining whether the amount of at least one of HER2 orp95 protein in the subject's sample is above a HER2 cutoff or a p95cutoff; and (d) indicating that the subject should receive treatmentwith a HER2-acting agent and a second form of cancer treatment if theamount of at least one of HER2 or p95 is above the HER2 cutoff or thep95 cutoff.

In another embodiment, the invention comprises a method for determiningan expected time to brain metastasis (TTBM) in a subject with aHER2-positive cancer comprising: (a) obtaining a biological sample of atumor from the subject's cancer; (b) measuring the amount of at leastone of HER2 or p95 in the biological sample; (c) determining whether theamount of at least one of HER2 or p95 protein in the subject's sample isabove a HER2 cutoff or a p95 cutoff; and (d) indicating the subject'sexpected TTBM based on the incidence of brain metastasis over time in areference population having HER2 or p95 levels above or below the HER2cutoff or p95 cutoff.

In an embodiment, the invention comprises a method of determining if asubject with HER2 positive cancer is within in a subset of HER2 positivecancer subjects that should be screened for brain metastasis,comprising: (a) obtaining a biological sample of a tumor from thesubject's cancer; (b) measuring the amount of at least one of HER2 orp95 in the biological sample; (c) determining whether the amount of atleast one of HER2 or p95 protein in the subject's sample is above a HER2cutoff or a p95 cutoff; and (d) indicating that the subject should bescreened for brain metastasis if the amount of at least one of HER2 orp95 is above the HER2 cutoff or the p95 cutoff.

Another embodiment is a method for predicting time to brain metastasis(TTBM) in a subject with a HER2-positive cancer comprising obtaining abiological sample of a tumor from the subject's cancer, (a) obtaining abiological sample of a tumor from the subject's cancer; (b) measuringthe amount of at least one of HER2 or p95 in the biological sample; (c)determining whether the amount of at least one of HER2 or p95 protein inthe subject's sample is above a HER2 cutoff or a p95 cutoff; and (d)identifying the TTBM based on the amount of HER2 or p95 protein in thebiological sample. In some embodiments, if the amount of HER2 in thebiological sample is above the HER2 cutoff the subject's chance of beingfree of brain metastasis is about 73% at about 1 year, about 61% atabout 2 years, and about 37% at about 3 years. In some embodiments, ifthe amount of HER2 in the biological sample is below the HER2 cutoff thesubject's chance of being free of brain metastasis is about 89% at about1 year, about 78% at about 2 years, and about 69% at about 3 years. Incertain embodiments, if the amount of p95 in the biological sample isabove the p95 cutoff the subject's chance of being free of brainmetastasis is about 77% at about 1 year, about 63% at about 2 years, andabout 40% at about 3 years. In some embodiments, if the amount of p95 inthe biological sample is below the p95 cutoff the subject's chance ofbeing free of brain metastasis is about 85% at about 1 year, about 77%at about 2 years, and about 67% at about 3 years. In certainembodiments, the amount of HER2 in the biological sample is below theHER2 cutoff and the amount of p95 is above the p95 cutoff the subject'schance of being free of brain metastasis is about 80% at about 1 year,about 66% at about 2 years, and about 50% at about 3 years. In certainembodiments, if the amount of HER2 in the biological sample is below theHER2 cutoff and the amount of p95 is below the p95 cutoff the subject'schance of being free of brain metastasis is about 94% at about 1 year,about 86% at about 2 years, and about 80% at about 3 years. In someembodiments, the subject has about a 2.6 fold increased risk of brainmetastasis if the amount of HER2 in the biological sample is above theHER2 cutoff as compared to if the amount is below the HER2 cutoff. Incertain embodiments, the subject has about a 2 fold increased risk ofbrain metastasis if the amount of p95 in the biological sample is abovethe p95 cutoff as compared to if the amount is below the p95 cutoff. Insome embodiments, the subject has about a 5.7 fold decreased risk ofbrain metastasis if the subject's cancer is Grade 1 or 2 and the amountof HER2 in the biological sample is below the HER2 cutoff as compared toif the subject's cancer was Grade 3 or if the subject's cancer was Grade1 or 2 and the amount of the amount of HER2 in the biological sample isabove the HER2 cutoff.

The embodiments set for below are aspects of each of the methodsdescribed herein.

In some embodiments, the subject's cancer has been characterized asHER2-positive based on elevated levels of HER2 gene expression, HER2protein level, or HER2 gene amplification. In some embodiments, thesubject's cancer comprises breast cancer. In certain embodiments, thesubject's cancer comprises primary breast cancer. In some embodiments,HER2 gene amplification is determined by fluorescence in situhybridization (FISH). In certain embodiments, the HER2 protein levelsare determined by an immunoassay. In certain embodiments, the HER2protein levels are determined by a VeraTag® assay. In some embodiments,HER2 gene amplification is determined by quantitation of HER2 mRNAlevels.

In certain embodiments, the subject has undergone treatment with a HER2acting agent that does not cross the blood-brain barrier. In someembodiments, the HER2-acting agent is a monoclonal antibody. In someembodiments, the monoclonal antibody is trastuzumab, pertuzumab,ertumaxomab, tratuzumab emtansine and/or MM-111. In some embodiments,the HER2-acting agent comprises a single-chain antibodies, an antibodyfragment (e.g., Fab fragment), or a genetically engineered protein thatcan bind to an antigen (e.g., an Affybody™, an Adnectin™, a mono-body, amodified Fc region fragment, an immuno-adhesin molecule, or other suchmolecules.

In some embodiments, the second form of cancer treatment comprises aHER2-targeted small molecule drug, chemotherapy and/or radiationtherapy. In certain embodiments, the HER2-targeted small molecule drugcomprises hydrophobic molecules such as, e.g., lapatinib, neratinib,AZD8931, ARRY-380, PF299, afatinib, pelitinib, S-222611, or AEE-788. Insome embodiments, other hydrophobic small molecule drugs areappropriate. In some embodiments, the second form of cancer treatmentcomprises a small molecule drug that binds to a protein that binds toHER2. For example, in some embodiments, the small molecule drugcomprises drugs that bind to HSP90 such as, e.g., 17-AAG or IPI-504.

The methods of the invention enable identification of quantitative HER2and p95 cutoffs to enable classification of subjects into risk subgroupsbased on the amount of HER2 and/or p95 in biological samples from thesubject. The methods of the invention also enable quantitativemeasurement of HER2 and/or p95 characterization of a subject's risk inrelation to a subject population as a whole as well.

For each of the methods disclosed herein, the method may furthercomprise the step of determining the HER2 cutoff and/or p95 cutoff. Insome embodiments, the HER2 cutoff and/or the p95 cutoff are determinedusing a VeraTag® assay. In certain embodiments, the HER2 cutoff and/orthe p95 cutoff are determined using other methods of quantitation knownin the art (e.g., mRNA quantitation, immunoassay, etc. as disclosedherein). In aspects of the invention, if the step of measuring theamount of HER2 protein in the biological sample and the step ofdetermining the HER2 cutoff both comprise use of a VeraTag® assay, theHER2 cutoff comprises a distinct and higher measure than the measureused to characterize the subject's cancer as HER2 positive.

In some embodiments, the HER2 cutoff comprises at least one of: (i) amedian amount of HER2 determined in a reference population of subjectswith HER2-positive breast cancer, or (ii) an optimized amount of HER2 asdetermined in a reference population of subjects with HER2-positivebreast cancer. In some embodiments, the HER2 cutoff is determined byVeraTag® assay. In certain embodiments, the optimized amount of HER2(the optimized cutoff) is 50 RF/mm². In certain embodiments, the HER2cutoff is 58 RF/mm². In some embodiments, the p95 cutoff comprises atleast one of: (i) a median amount of p95 determined in a referencepopulation of subjects with HER2-positive breast cancer, or (ii) anoptimized amount of p95 as determined in a reference population ofsubjects with HER2-positive breast cancer. In some embodiments, the p95cutoff is determined by VeraTag® assay. In certain embodiments, theoptimized amount of p95 (the optimized cutoff) is 2.8 RF/mm². In someembodiments, the reference population of subjects with HER2-positivebreast cancer have undergone treatment with a HER2-acting agent thatdoes not cross the blood-brain barrier.

In certain embodiments, the amounts of HER2 protein present aredetermined by contacting a biological sample from a subject with cancerwith a binding compound having a molecular tag attached thereto by acleavable linkage and a cleaving probe having a cleavage inducing-moietyand detecting whether and what molecular tag is released. Someembodiments may be referred to as a HERmark® assay (see e.g., FIGS. 1Aand 2A). The HERmark® assay uses two monoclonal antibodies specific fordifferent unique epitopes on the HER2 protein. This enables bothantibodies to bind to the same HER2 receptor in close proximity. Thefluorescent VeraTag® reporter molecule (“Tag”) is conjugated to amonoclonal antibody specific for HER2. A second HER2-specific monoclonalantibody is conjugated to biotin, which is then linked to aphotosensitizer molecule (PM). Photoactivation of the sample at aspecific wavelength activates the PM, generating singlet oxygen. Thesinglet oxygen can cleave the VeraTag® reporter in close proximity. Seee.g., FIG. 2C. The released VeraTag® reporter is collected andsubsequently quantified using standard capillary electrophoresis. Theamount of cleaved VeraTag® reporter is proportional to the concentrationof HER2 in the sample.

In certain embodiments, the binding compound and the cleaving probe eachspecifically binds HER2. In certain embodiments, the cleaving probe andthe binding probe do not both bind the same epitope. See e.g., FIGS. 1Aand 2A. In some embodiments, the cleaving probe and the binding probeboth bind the same epitope. See e.g., FIG. 2B. In certain embodiments,if the binding compound is within an effective proximity of thecleavage-inducing moiety of the cleaving probe, the cleavage-inducingmoiety cleaves the cleavable linker so that the molecular tag isreleased. In some embodiments, the amount of HER2 in a sample isdetermined using a first binding compound specific for HER2 and secondbinding compound specific for the first binding compound, wherein thesecond binding compound comprises one or more molecular probes attachedthereto. In certain embodiments, the molecular probes are attached via acleavable linkage. In some embodiments, the cleavable linkage is cleavedby a reducing agent. For example, in some embodiments, the reducingagent comprises dithiothreitol (DTT). See e.g., FIG. 1B. Examples ofdetection of HER2 by an assay for detection of total HER2 and/or HER2homodimers is provided in commonly owned U.S. Patent ApplicationPublication Nos. 2009/0191559, 2010/0143927, 2010/0210034, and2010/0233732, which are incorporated by reference in their entiretiesherein.

In certain embodiments, the amounts of p95 in a sample are determinedusing a proximity probe that is capable of binding p95 or an analytewhich binds p95 or a p95 complex, the proximity probe having aneffective proximity, and having one or more molecular probes attached,wherein binding of the proximity probe and binding compound within theeffective proximity produces a signal from the molecular probes thatcorrelates with the presence and/or quantity of p95 or p95 complex. Theproximity probe and/or binding compound may further comprise anantibody.

In some embodiments, the amounts of p95 in a sample are determined usinga first binding compound specific for p95 but not full length HER2 andsecond binding compound specific for the first binding compound, whereinthe second binding compound comprises one or more molecular probesattached thereto. In certain embodiments, the molecular probes areattached via a cleavable linkage. In some embodiments, the cleavablelinkage is cleaved by a reducing agent. For example, in someembodiments, the reducing agent comprises dithiothreitol (DTT). Seee.g., FIG. 1B. Examples of detection of p95 is provided in commonlyowned U.S. Patent Application Publication Nos. 2010/0143927 and2010/0210034, now issued as U.S. Pat. Nos. 8,470,542 and 8,349,574,respectively. In certain embodiments, the first binding compoundspecific for p95 comprises or is one of the antibodies produced by ahybridoma cell lines having accession number PTA-9738 (p95.D3.4),PTA-9739 (p95.D8.2) and PTA-9740 (p95.D9.1), as described in U.S. PatentApplication Publication Nos. 2010/0143927 and 2010/0210034, anddeposited on Jan. 28, 2009, to the American Type Culture Collection(ATCC, 10801 University 45 Blvd., Manassas, Va.) under conditionsprescribed by the Budapest Treaty.

Proximity assays are increasingly useful for the understanding of thebiological role of molecular complexes, as well as in the study ofbiomarkers. For example, binding compounds that specifically bind atarget protein can be coupled with many different detection systems tomeasure the presence and/or quantity of the target protein. Any methodknown to one of skill in the art to be useful for determining an amountof a target protein can be used in accordance with the presentinvention. Such methods include but are not limited to Foersterresonance energy transfer (FRET), bioluminescence resonance energytransfer (BRET), biomolecular fluorescence complementation, proximityligation assay (PLA), scintillation proximity assay (SPA), andimmunoassays with target protein specific antibodies, including, e.g.,VeraTag® assays, or any other method that is well known to one skilledin the art.

Many advantages are provided by measuring HER2 and p95 using releasablemolecular tags, including separation of released molecular tags from anassay mixture providing greatly reduced background and a significantgain in sensitivity and separation and detection providing a convenientmultiplexing capability so that multiple receptor complex components maybe readily measured simultaneously in the same assay. Assays employingsuch tags can have a variety of forms and are disclosed in the followingreferences: U.S. Pat. Nos. 7,105,308; 6,627,400; 7,402,397; 7,402,398and 7,402,399, as well as International Patent Publication No. WO2004/011900, each of which is incorporated herein by reference in theirentirety. A wide variety of separation techniques may be employed thatcan distinguish molecules based on one or more physical, chemical oroptical differences among molecules being separated includingelectrophoretic mobility, molecular weight, shape, solubility, pKa,hydrophobicity, charge, charge/mass ratio or polarity. In oneembodiment, molecular tags in a plurality or set differ inelectrophoretic mobility and optical detection characteristics and areseparated by electrophoresis. In another embodiment, molecular tags in aplurality or set may differ in molecular weight, shape, solubility, pKa,hydrophobicity, charge, polarity and are separated by normal phase orreverse phase HPLC, ion exchange HPLC, capillary electrochromatography,mass spectroscopy or gas phase chromatography.

Sets of molecular tags are provided that can be separated into distinctbands or peaks by a separation technique after they are released frombinding compounds. Identification and quantification of such peaksprovides a measure or profile of the presence and/or amounts of p95.Molecular tags within a set may be chemically diverse; however, forconvenience, sets of molecular tags are usually chemically related. Forexample, they may all be peptides or they may consist of differentcombinations of the same basic building blocks or monomers or they maybe synthesized using the same basic scaffold with different substituentgroups for imparting different separation characteristics. The number ofmolecular tags in a plurality may vary depending on several factorsincluding the mode of separation employed, the labels used on themolecular tags for detection, the sensitivity of the binding moietiesand the efficiency with which the cleavable linkages are cleaved.

The invention relates to HER acting agents. For example, a HER2-actingagent can be any such agent known to one of skill in the art. In certainembodiments, the HER2-acting agent is selected from the group consistingof pertuzumab, trastuzumab, ertumaxomab, 17-AAG, IPI-504, neratinib,AZD8931, ARRY-380, PF299, afatinib, pelitinib, S-222611 AEE-788 andlapatinib. In a preferred embodiment, the HER2-acting agent istrastuzumab (Herceptin®). See, e.g., Goldenberg, 1999, Clin Ther.21:309-18; and Shak, 1999, Semin Oncol. 26:71-7. Also, other HER2 actingagents may be evaluated using the methods described herein.

Samples containing HER2 and/or HER2 homodimers suitable for use asbiomarkers may come from a wide variety of sources, including cellcultures, animal or plant tissues, patient biopsies, or the like.Preferably, samples are human patient samples. Samples are prepared forassays of the invention using conventional techniques, which may dependon the source from which a sample is taken. For biopsies and medicalspecimens, guidance is provided in the following references: Theory andPractice of Histological Techniques, 1977 (Bancroft J D & Stevens A,eds.), Churchill Livingstone, Edinburgh; Pearse, 1980, Histochemistry.Theory and applied. 4^(th) ed., Churchill Livingstone, Edinburgh.

In the area of cancerous disease status, examples of patient tissuesamples that may be used include, but are not limited to, breast,prostate, ovary, colon, lung, endometrium, stomach, salivary gland, orpancreas. The tissue sample can be obtained by a variety of proceduresincluding surgical excision, aspiration, or biopsy. The tissue may befresh or frozen. In one embodiment, assays of the invention are carriedout on tissue samples that have been fixed and embedded in paraffin anda step of deparaffination is be carried out. A tissue sample may befixed (i.e., preserved) by conventional methodology. See, e.g., Manualof Histological Staining Method of the Armed Forces Institute ofPathology, 1960, 3^(rd) edition (Lee G. Luna, HT (ASCP) ed.), TheBlakston Division McGraw-Hill Book Company, New York; The Armed ForcesInstitute of Pathology Advanced Laboratory Methods in Histology andPathology, 1994 (Ulreka V. Mikel, ed.), Armed Forces Institute ofPathology, American Registry of Pathology, Washington, D.C. One of skillin the art will appreciate that the choice of a fixative is determinedby the purpose for which the tissue is to be histologically stained orotherwise analyzed. One of skill in the art will also appreciate thatthe length of fixation depends upon the size of the tissue sample andthe fixative used.

Generally, a tissue sample is first fixed and is then dehydrated throughan ascending series of alcohols, infiltrated, and embedded with paraffinor other sectioning media so that the tissue sample may be sectioned.Alternatively, one may section the tissue and fix the sections obtained.By way of example, the tissue sample may be embedded and processed inparaffin by conventional methodology according to conventionaltechniques described by the references provided above. Examples ofparaffin that may be used include, but are not limited to, Paraplast®,Broloid®, and Tissuemay®. Once the tissue sample is embedded, the samplemay be sectioned by a microtome according to conventional techniques.Sections may have a thickness in a range from about three microns toabout twelve microns, and preferably, a thickness in a range of fromabout 5 microns to about 10 microns. In one aspect, a section may have asurface area of from about 10 mm² to about 1 cm². Once cut, the sectionsmay be attached to slides by several standard methods. Examples of slideadhesives include, but are not limited to, silane, gelatin andpoly-L-lysine. Paraffin embedded sections may be attached to positivelycharged slides and/or slides coated with poly-L-lysine.

If paraffin has been used as the embedding material, the tissue sectionsare generally deparaffinized and rehydrated to water prior to detectionof biomarkers. Tissue sections may be deparaffinized by severalconventional standard methodologies. For example, xylenes and agradually descending series of alcohols may be used according toconventional techniques described by the references provided above.Alternatively, commercially available deparaffinizing non-organic agentssuch as Hemo-De® (CMS, Houston, Tex.) may be used.

Mammalian tissue culture cells, or fresh or frozen tissues may beprepared by conventional cell lysis techniques (e.g., 0.14 M NaCl, 1.5mM MgCl₂, 10 mM Tris-Cl (pH 8.6), 0.5% Nonidet P-40, and protease and/orphosphatase inhibitors as required). For fresh mammalian tissues, samplepreparation may also include a tissue disaggregation step, such ascrushing, mincing, grinding or sonication.

Many advantages are provided by measuring dimer populations usingreleasable molecular tags, including (1) separation of releasedmolecular tags from an assay mixture provides greatly reduced backgroundand a significant gain in sensitivity; and (2) the use of molecular tagsthat are specially designed for ease of separation and detectionprovides a convenient multiplexing capability so that multiple receptorcomplex components may be readily measured simultaneously in the sameassay. Assays employing such tags can have a variety of forms and aredisclosed in the following references: U.S. Pat. Nos. 6,627,400,6,673,550, 6,949,347, 7,105,308; published U.S. Patent Application No.and 2009/0191559; and International Patent Publication No. WO2004/011900, each of which are incorporated herein by reference in theirentireties. For example, a wide variety of separation techniques may beemployed that can distinguish molecules based on one or more physical,chemical or optical differences among molecules being separatedincluding electrophoretic mobility, molecular weight, shape, solubility,pKa, hydrophobicity, charge, charge/mass ratio, or polarity. In oneaspect, molecular tags in a plurality or set differ in electrophoreticmobility and optical detection characteristics and are separated byelectrophoresis. In another aspect, molecular tags in a plurality or setmay differ in molecular weight, shape, solubility, pKa, hydrophobicity,charge, or polarity and are separated by normal phase or reverse phaseHPLC, ion exchange HPLC, capillary electrochromatography, massspectroscopy, or gas phase chromatography.

Sets of molecular tags are provided that can be separated into distinctbands or peaks by a separation technique after they are released frombinding compounds. Identification and quantification of such peaksprovides a measure or profile of the presence and/or amounts of receptordimers. Molecular tags within a set may be chemically diverse; however,for convenience, sets of molecular tags are usually chemically related.For example, they may all be peptides or they may consist of differentcombinations of the same basic building blocks or monomers or they maybe synthesized using the same basic scaffold with different substituentgroups for imparting different separation characteristics. The number ofmolecular tags in a plurality may vary depending on several factorsincluding the mode of separation employed, the labels used on themolecular tags for detection, the sensitivity of the binding moietiesand the efficiency with which the cleavable linkages are cleaved.

Measurements made directly on tissue samples may be normalized byincluding measurements on cellular or tissue targets that arerepresentative of the total cell number in the sample and/or the numbersof particular subtypes of cells in the sample. The additionalmeasurement may be preferred, or even necessary, because of the cellularand tissue heterogeneity in patient samples, particularly tumor samples,which may comprise substantial fractions of normal cells.

As mentioned above, mixtures containing pluralities of different bindingcompounds may be provided, wherein each different binding compound hasone or more molecular tags attached through cleavable linkages. Thenature of the binding compound, cleavable linkage and molecular tag mayvary widely. A binding compound may comprise an antibody bindingcomposition, an antibody, a peptide, a peptide or non-peptide ligand fora cell surface receptor, a protein, an oligonucleotide, anoligonucleotide analog, such as a peptide nucleic acid, a lectin or anyother molecular entity that is capable of specifically binding to atarget protein or molecule or stable complex formation with an analyteof interest, such as a HER2 homodimer. In one aspect, a binding compoundcan be represented by the following formula:B-(L-E)_(k)wherein B is binding moiety; L is a cleavable linkage and E is amolecular tag. In homogeneous assays, cleavable linkage, L, may be anoxidation-labile linkage, and more preferably, it is a linkage that maybe cleaved by singlet oxygen. The moiety “-(L-E)_(k)” indicates that asingle binding compound may have multiple molecular tags attached viacleavable linkages. In one aspect, k is an integer greater than or equalto one, but in other embodiments, k may be greater than several hundred,e.g. 100 to 500 or k is greater than several hundred to as many asseveral thousand, e.g. 500 to 5000. Usually each of the plurality ofdifferent types of binding compounds has a different molecular tag, E.Cleavable linkages, e.g. oxidation-labile linkages, and molecular tags,E, are attached to B by way of conventional chemistries.

Preferably, B is an antibody binding composition that specifically bindsto a target, such as an antigenic determinant on HER2. Antibodiesspecific for HER2 epitopes are provided in the examples set forthherein. Antibody compositions are readily formed from a wide variety ofcommercially available antibodies, either monoclonal or polyclonal. Inparticular, antibodies specific for epidermal growth factor receptorsare disclosed in U.S. Pat. Nos. 5,677,171; 5,772,997; 5,968,511;5,480,968; and 5,811,098, each of which is incorporated by reference inits entirety. U.S. Pat. No. 5,599,681, hereby also incorporated byreference in its entirety, discloses antibodies specific forphosphorylation sites of proteins. Commercial vendors, such as CellSignaling Technology (Beverly, Mass.), Biosource International(Camarillo, Calif.) and Upstate (Charlottesville, Va.) also providemonoclonal and polyclonal antibodies.

Cleavable linkage, L, can be virtually any chemical linking group thatmay be cleaved under conditions that do not degrade the structure oraffect detection characteristics of the released molecular tag, E.Whenever a cleaving probe is used in a homogeneous assay format,cleavable linkage, L, is cleaved by a cleavage agent generated by thecleaving probe that acts over a short distance so that only cleavablelinkages in the immediate proximity of the cleaving probe are cleaved.Typically, such an agent must be activated by making a physical orchemical change to the reaction mixture so that the agent produces ashort lived active species that diffuses to a cleavable linkage toeffect cleavage. In a homogeneous format, the cleavage agent ispreferably attached to a binding moiety, such as an antibody, thattargets prior to activation the cleavage agent to a particular site inthe proximity of a binding compound with releasable molecular tags. Insuch embodiments, a cleavage agent is referred to herein as a“cleavage-inducing moiety.” An exemplary cleavage linkage is illustratedin FIG. 2C.

In a non-homogeneous format, because specifically bound bindingcompounds are separated from unbound binding compounds, a widerselection of cleavable linkages and cleavage agents are available foruse. Cleavable linkages may not only include linkages that are labile toreaction with a locally acting reactive species, such as hydrogenperoxide, singlet oxygen, or the like, but also linkages that are labileto agents that operate throughout a reaction mixture, such asbase-labile linkages, photocleavable linkages, linkages cleavable byreduction, linkages cleaved by oxidation, acid-labile linkages, andpeptide linkages cleavable by specific proteases. References describingmany such linkages include Greene and Wuts, 1991, Protective Groups inOrganic Synthesis, Second Edition, John Wiley & Sons, New York;Hermanson, 1996, Bioconjugate Techniques, Academic Press, New York; andU.S. Pat. No. 5,565,324.

In one aspect, commercially available cleavable reagent systems may beemployed with the invention. For example, a disulfide linkage may beintroduced between an antibody binding composition and a molecular tagusing a heterofunctional agent such as N-succinimidyl3-(2-pyridyldithio)propionate (SPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene (SMPT) orthe like, available from vendors such as Pierce Chemical Company(Rockford, Ill.). Disulfide bonds introduced by such linkages can bebroken by treatment with a reducing agent, such as dithiothreitol (DTT),dithioerythritol (DTE), 2-mercaptoethanol or sodium borohydride. Typicalconcentrations of reducing agents to effect cleavage of disulfide bondsare in the range of from 10 to 100 mM. An oxidatively labile linkage maybe introduced between an antibody binding composition and a moleculartag using the homobifunctional NHS ester cross-linking reagent,disuccinimidyl tartarate (DST) (available from Pierce) that containscentral cis-diols that are susceptible to cleavage with sodium periodate(e.g., 15 mM periodate at physiological pH for 4 hours). Linkages thatcontain esterified spacer components may be cleaved with strongnucleophilic agents, such as hydroxylamine, e.g., 0.1 N hydroxylamine,pH 8.5, for 3-6 hours at 37° C. Such spacers can be introduced by ahomobifunctional cross-linking agent such as ethylene glycolbis(succinimidylsuccinate) (EGS) available from Pierce (Rockford, Ill.).A base labile linkage can be introduced with a sulfone group.Homobifunctional cross-linking agents that can be used to introducesulfone groups in a cleavable linkage includebis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES), and4,4-difluoro-3,3-dinitrophenylsulfone (DFDNPS). Exemplary basicconditions for cleavage include 0.1 M sodium phosphate, adjusted to pH11.6 by addition of Tris base, containing 6 M urea, 0.1% SDS, and 2 mMDTT, with incubation at 37° C. for 2 hours. Photocleavable linkages alsoinclude those disclosed in U.S. Pat. No. 5,986,076.

When L is oxidation labile, L may be a thioether or its selenium analog;or an olefin, which contains carbon-carbon double bonds, whereincleavage of a double bond to an oxo group, releases the molecular tag,E. Illustrative oxidation labile linkages are disclosed in U.S. Pat.Nos. 5,622,929, 6,627,400 and 6,949,347; each of which is herebyincorporated by reference in their entirety.

Molecular tag, E, in the present invention may comprise an electrophorictag as described in the following references when separation ofpluralities of molecular tags are carried out by gas chromatography ormass spectrometry: See, e.g., Zhang et al., 2002, Bioconjugate Chem.13:1002-1012; Giese, 1983, Anal. Chem. 2:165-168; and U.S. Pat. Nos.4,650,750; 5,360,819; 5,516,931; and 5,602,273, each of which is herebyincorporated by reference in their entirety.

Molecular tag, E, is preferably a water-soluble organic compound that isstable with respect to the active species, especially singlet oxygen,and that includes a detection or reporter group. Otherwise, E may varywidely in size and structure. In one aspect, E has a molecular weight inthe range of from about 50 to about 2500 daltons, more preferably, fromabout 50 to about 1500 daltons. E may comprise a detection group forgenerating an electrochemical, fluorescent or chromogenic signal. Inembodiments employing detection by mass, E may not have a separatemoiety for detection purposes. Preferably, the detection group generatesa fluorescent signal. An exemplary molecular tag (Pro11) is shown inFIG. 2.

Molecular tags within a plurality are selected so that each has a uniqueseparation characteristic and/or a unique optical property with respectto the other members of the same plurality. In one aspect, thechromatographic or electrophoretic separation characteristic isretention time under a set of standard separation conditionsconventional in the art, e.g., voltage, column pressure, column type,mobile phase, or electrophoretic separation medium. In another aspect,the optical property is a fluorescence property, such as emissionspectrum, fluorescence lifetime, or fluorescence intensity at a givenwavelength or band of wavelengths. Preferably, the fluorescence propertyis fluorescence intensity. For example, each molecular tag of aplurality may have the same fluorescent emission properties, but eachwill differ from one another by virtue of a unique retention time. Onthe other hand, one or two or more of the molecular tags of a pluralitymay have identical migration or retention times, but they will haveunique fluorescent properties, e.g. spectrally resolvable emissionspectra, so that all the members of the plurality are distinguishable bythe combination of molecular separation and fluorescence measurement.

Preferably, released molecular tags are detected by electrophoreticseparation and the fluorescence of a detection group. In suchembodiments, molecular tags having substantially identical fluorescenceproperties have different electrophoretic mobilities so that distinctpeaks in an electropherogram are formed under separation conditions.Preferably, pluralities of molecular tags of the invention are separatedby conventional capillary electrophoresis apparatus, either in thepresence or absence of a conventional sieving matrix. During or afterelectrophoretic separation, the molecular tags are detected oridentified by recording fluorescence signals and migration times (ormigration distances) of the separated compounds or by constructing achart of relative fluorescent and order of migration of the moleculartags (e.g., as an electropherogram). Preferably, the presence, absenceand/or amounts of molecular tags are measured by using one or morestandards as disclosed by published U.S. Patent Application No.2003/0170734, which is hereby incorporated by reference in its entirety.

Pluralities of molecular tags may also be designed for separation bychromatography based on one or more physical characteristics thatinclude molecular weight, shape, solubility, pKa, hydrophobicity,charge, polarity or the like, e.g. as disclosed in published U.S. PatentApplication No. 2003/0235832, which hereby is incorporated by referencein its entirety. A chromatographic separation technique is selectedbased on parameters such as column type, solid phase, mobile phase andthe like, followed by selection of a plurality of molecular tags thatmay be separated to form distinct peaks or bands in a single operation.Several factors determine which HPLC technique is selected for use inthe invention, including the number of molecular tags to be detected(i.e., the size of the plurality), the estimated quantities of eachmolecular tag that will be generated in the assays, the availability andease of synthesizing molecular tags that are candidates for a set to beused in multiplexed assays, the detection modality employed and theavailability, robustness, cost and ease of operation of HPLCinstrumentation, columns and solvents. Generally, columns and techniquesare favored that are suitable for analyzing limited amounts of sampleand that provide the highest resolution separations. Guidance for makingsuch selections can be found in the literature, such as, for example,Snyder et al., 1988, Practical HPLC Method Development, John Wiley &Sons, New York; Millner, 1999, High Resolution Chromatography: APractical Approach, Oxford University Press, New York; Chi-San Wu, 1999,Column Handbook for Size Exclusion Chromatography, Academic Press, SanDiego; and Oliver, 1989, HPLC of Macromolecules: A Practical Approach,Oxford University Press, Oxford, England.

In one aspect, molecular tag, E, is (M, D), where M is amobility-modifying moiety and D is a detection moiety. The notation “(M,D)” is used to indicate that the ordering of the M and D moieties may besuch that either moiety can be adjacent to the cleavable linkage, L.That is, “B-L-(M, D)” designates binding compound of either of twoforms: “B-L-M-D” or “B-L-D-M.”

Detection moiety, D, may be a fluorescent label or dye, a chromogeniclabel or dye or an electrochemical label. Preferably, D is a fluorescentdye. Exemplary fluorescent dyes for use with the invention includewater-soluble rhodamine dyes, fluoresceins, 4,7-dichlorofluoresceins,benzoxanthene dyes and energy transfer dyes, as disclosed in thefollowing references: Handbook of Molecular Probes and ResearchReagents, 8^(th) ed., 2002, Molecular Probes, Eugene, Oreg.; U.S. Pat.Nos. 4,318,846, 4,997,928, 5,945,526, 6,096,723, 6,191,278, and6,372,907, and Lee et al., 1997, Nucleic Acids Research 25:2816-2822.Preferably, D is a fluorescein or a fluorescein derivative.

Once each of the binding compounds is separately derivatized by adifferent molecular tag, it is pooled with other binding compounds toform a plurality of binding compounds. Usually, each different kind ofbinding compound is present in a composition in the same proportion;however, proportions may be varied as a design choice so that one or asubset of particular binding compounds are present in greater or lowerproportion depending on the desirability or requirements for aparticular embodiment or assay. Factors that may affect such designchoices include, but are not limited to, antibody affinity and avidityfor a particular target, relative prevalence of a target, fluorescentcharacteristics of a detection moiety of a molecular tag and the like.

A cleavage-inducing moiety, or cleaving agent, is a group that producesan active species that is capable of cleaving a cleavable linkage,preferably by oxidation. Preferably, the active species is a chemicalspecies that exhibits short-lived activity so that its cleavage-inducingeffects are only in the proximity of the site of its generation. Eitherthe active species is inherently short lived, so that it will not createsignificant background beyond the proximity of its creation, or ascavenger is employed that efficiently scavenges the active species, sothat it is not available to react with cleavable linkages beyond a shortdistance from the site of its generation. Illustrative active speciesinclude singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals,phenoxy radical, superoxide and the like. Illustrative quenchers foractive species that cause oxidation include polyenes, carotenoids,vitamin E, vitamin C, amino acid-pyrrole N-conjugates of tyrosine,histidine and glutathione. See, e.g. Beutner et al., 2000, Meth.Enzymol. 319:226-241.

One consideration in designing assays employing a cleavage-inducingmoiety and a cleavable linkage is that they not be so far removed fromone another when bound to a receptor complex that the active speciesgenerated by the cleavage-inducing moiety cannot efficiently cleave thecleavable linkage. In one aspect, cleavable linkages preferably arewithin about 1000 nm and preferably within about 20-200 nm, of a boundcleavage-inducing moiety. More preferably, for photosensitizercleavage-inducing moieties generating singlet oxygen, cleavable linkagesare within about 20-100 nm of a photosensitizer in a receptor complex.The range within which a cleavage-inducing moiety can effectively cleavea cleavable linkage (that is, cleave enough molecular tag to generate adetectable signal) is referred to herein as its “effective proximity.”One of ordinary skill in the art will recognize that the effectiveproximity of a particular sensitizer may depend on the details of aparticular assay design and may be determined or modified by routineexperimentation.

A sensitizer is a compound that can be induced to generate a reactiveintermediate, or species, usually singlet oxygen. Preferably, asensitizer used in accordance with the invention is a photosensitizer.Other sensitizers included within the scope of the invention arecompounds that on excitation by heat, light, ionizing radiation orchemical activation will release a molecule of singlet oxygen. The bestknown members of this class of compounds include the endoperoxides suchas 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Further sensitizers aredisclosed by Di Mascio et al., 1994, FEBS Lett. 355:287; and Kanofsky,1983, J. Biol. Chem. 258:5991-5993; Pierlot et al., 2000, Meth. Enzymol.319:3-20.

Photosensitizers may be attached directly or indirectly, via covalent ornon-covalent linkages, to the binding agent of a class-specific reagent.Guidance for constructing such compositions, particularly for antibodiesas binding agents are available in the literature, e.g. in the fields ofphotodynamic therapy, immunodiagnostics, and the like. Exemplaryguidance may be found in Ullman et al., 1994, Proc. Natl. Acad. Sci. USA91, 5426-5430; Strong et al., 1994, Ann. New York Acad. Sci. 745:297-320; Yarmush et al., 1993, Crit. Rev. Therapeutic Drug Carrier Syst.10: 197-252; and U.S. Pat. Nos. 5,340,716, 5,516,636, 5,709,994, and6,251,581.

A large variety of light sources are available to photo-activatephotosensitizers to generate singlet oxygen. Both polychromatic andmonochromatic sources may be used as long as the source is sufficientlyintense to produce enough singlet oxygen in a practical time duration.The length of the irradiation depends on the nature of thephotosensitizer, the nature of the cleavable linkage, the power of thesource of irradiation and its distance from the sample. In general, theperiod for irradiation may be less than about a microsecond to as longas about 10 minutes, usually in the range of about one millisecond toabout 60 seconds. The intensity and length of irradiation should besufficient to excite at least about 0.1% of the photosensitizermolecules, usually at least about 30% of the photosensitizer moleculesand preferably, substantially all of the photosensitizer molecules.Exemplary light sources include lasers such as, e.g., helium-neonlasers, argon lasers, YAG lasers, He/Cd lasers and ruby lasers;photodiodes; mercury, sodium and xenon vapor lamps; incandescent lampssuch as, e.g., tungsten and tungsten/halogen and flashlamps. Anexemplary photoactivation device suitable for use in the methods of theinvention is disclosed International Patent Publication No. WO03/051669. In such embodiments, the photoactivation device is an arrayof light emitting diodes (LEDs) mounted in housing that permits thesimultaneous illumination of all the wells in a 96-well plate.

Examples of photosensitizers that may be utilized in the presentinvention are those that have the above properties and those disclosedby U.S. Pat. Nos. 5,536,834, 5,763,602, 5,565,552, 5,709,994, 5,340,716,5,516,636, 6,251,581, and 6,001,673; published European PatentApplication No. 0484027; Martin et al., 1990, Methods Enzymol.186:635-645; and Yarmush et al., 1993, Crit. Rev. Therapeutic DrugCarrier Syst. 10:197-252. As with sensitizers, in certain embodiments, aphotosensitizer may be associated with a solid phase support by beingcovalently or non-covalently attached to the surface of the support orincorporated into the body of the support. In general, thephotosensitizer is associated with the support in an amount necessary toachieve the necessary amount of singlet oxygen. Generally, the amount ofphotosensitizer is determined empirically according to routine methods.

In one embodiment, a photosensitizer is incorporated into a latexparticle to form photosensitizer beads, e.g. as disclosed by U.S. Pat.Nos. 5,709,994 and 6,346,384; and International Patent Publication No.WO 01/84157. Alternatively, photosensitizer beads may be prepared bycovalently attaching a photosensitizer, such as rose bengal, to 0.5micron latex beads by means of chloromethyl groups on the latex toprovide an ester linking group, as described in J. Amer. Chem. Soc.,97:3741 (1975). This reaction may be carried out, for example, in aconventional 96-well or 384-well microtiter plate, or the like, having afilter membrane that forms one wall, e.g. the bottom, of the wells thatallows reagents to be removed by the application of a vacuum. Thisallows the convenient exchange of buffers, if the buffer required forspecific binding of binding compounds is different than the bufferrequired for either singlet oxygen generation or separation. Forexample, in the case of antibody-based binding compounds, a high saltbuffer is required. If electrophoretic separation of the released tagsis employed, then better performance is achieved by exchanging thebuffer for one that has a lower salt concentration suitable forelectrophoresis.

As an example, a cleaving probe may comprise a primary haptenatedantibody and a secondary anti-hapten binding protein derivatized withmultiple photosensitizer molecules. A preferred primary haptenatedantibody is a biotinylated antibody and preferred secondary anti-haptenbinding proteins may be either an anti-biotin antibody or streptavidin.Other combinations of such primary and secondary reagents are well knownin the art. Exemplary combinations of such reagents are taught byHaugland, 2002, Handbook of Fluorescent Probes and Research Reagents,Ninth Edition, Molecular Probes, Eugene, Oreg. An exemplary combinationof such reagents is described below. There binding compounds havingreleasable tags (“mT₁” and “mT₂”), and primary antibody derivatized withbiotin are specifically bound to different epitopes of receptor dimer inmembrane. Biotin-specific binding protein, e.g. streptavidin, isattached to biotin bringing multiple photosensitizers into effectiveproximity of binding compounds. Biotin-specific binding protein may alsobe an anti-biotin antibody and photosensitizers may be attached via freeamine group on the protein by conventional coupling chemistries, e.g.,Hermanson (supra). An exemplary photosensitizer for such use is an NHSester of methylene blue prepared as disclosed in published EuropeanPatent Application 0510688.

The following general discussion of methods and specific conditions andmaterials are by way of illustration and not limitation. One of skill inthe art will understand how the methods described herein can be adaptedto other applications, particularly with using different samples, celltypes and target complexes.

In conducting the methods of the invention, a combination of the assaycomponents is made, including the sample being tested, the bindingcompounds and optionally the cleaving probe. Generally, assay componentsmay be combined in any order. In certain applications, however, theorder of addition may be relevant. For example, one may wish to monitorcompetitive binding, such as in a quantitative assay. Or one may wish tomonitor the stability of an assembled complex. In such applications,reactions may be assembled in stages.

The amounts of each reagent can generally be determined empirically. Theamount of sample used in an assay will be determined by the predictednumber of target complexes present and the means of separation anddetection used to monitor the signal of the assay. In general, theamounts of the binding compounds and the cleaving probe can be providedin molar excess relative to the expected amount of the target moleculesin the sample, generally at a molar excess of at least about 1.5, moredesirably about 10-fold excess, or more. In specific applications, theconcentration used may be higher or lower, depending on the affinity ofthe binding agents and the expected number of target molecules presenton a single cell. Where one is determining the effect of a chemicalcompound on formation of oligomeric cell surface complexes, the compoundmay be added to the cells prior to, simultaneously with or afteraddition of the probes, depending on the effect being monitored.

The assay mixture can be combined and incubated under conditions thatprovide for binding of the probes to the cell surface molecules, usuallyin an aqueous medium, generally at a physiological pH (comparable to thepH at which the cells are cultures), maintained by a buffer at aconcentration in the range of about 10 to 200 mM. Conventional buffersmay be used, as well as other conventional additives as necessary, suchas salts, growth medium, stabilizers, etc. Physiological and constanttemperatures are normally employed. Incubation temperatures normallyrange from about 4° to 70° C., usually from about 15° to 45° C., moreusually about 25° to 37° C.

After assembly of the assay mixture and incubation to allow the probesto bind to cell surface molecules, the mixture can be treated toactivate the cleaving agent to cleave the tags from the bindingcompounds that are within the effective proximity of the cleaving agent,releasing the corresponding tag from the cell surface into solution. Thenature of this treatment will depend on the mechanism of action of thecleaving agent. For example, where a photosensitizer is employed as thecleaving agent, activation of cleavage can comprise irradiation of themixture at the wavelength of light appropriate to the particularsensitizer used.

Following cleavage, the sample can then be analyzed to determine theidentity of tags that have been released. Where an assay employing aplurality of binding compounds is employed, separation of the releasedtags will generally precede their detection. The methods for bothseparation and detection are determined in the process of designing thetags for the assay. A preferred mode of separation employselectrophoresis, in which the various tags are separated based on knowndifferences in their electrophoretic mobilities.

As mentioned above, in some embodiments, if the assay reactionconditions may interfere with the separation technique employed, it maybe necessary to remove, or exchange, the assay reaction buffer prior tocleavage and separation of the molecular tags. For example, assayconditions may include salt concentrations (e.g. required for specificbinding) that degrade separation performance when molecular tags areseparated on the basis of electrophoretic mobility. Thus, such high saltbuffers may be removed, e.g., prior to cleavage of molecular tags, andreplaced with another buffer suitable for electrophoretic separationthrough filtration, aspiration, dilution or other means.

In certain embodiments, the subject may be administered a combinationtherapy that includes a HER2-acting agent. In some embodiments, theHER2-acting agent may be trastuzumab. The combination therapy caninclude trastuzumab in combination with one or more of anychemotherapeutic agent known to one of skill in the art withoutlimitation. Preferably, the chemotherapeutic agent has a differentmechanism of action from trastuzumab. For example, the chemotherapeuticagent can be an anti-metabolite (e.g., 5-flourouricil (5-FU),methotrexate (MTX), fludarabine, etc.), an antimicrotubule agent (e.g.,vincristine; vinblastine; taxanes such as paclitaxel and docetaxel;etc.), an alkylating agent (e.g., cyclophosphamide, melphalan,bischloroethylnitrosurea, etc.), platinum agents (e.g., cisplatin,carboplatin, oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g.,doxorubicin, daunorubicin, etc.), antibiotic agents (e.g., mitomycin-C,actinomycin D, etc.), topoisomerase inhibitors (e.g., etoposide,camptothecins, etc.) or other any other chemotherapeutic agents known toone skilled in the art.

Particular examples of chemotherapeutic agents that can be used in thevarious embodiments of the invention, including pharmaceuticalcompositions, dosage forms, and kits of the invention, include, withoutlimitation, cytarabine, melphalan, topotecan, fludarabine, etoposide,idarubicin, daunorubicin, mitoxantrone, cisplatin paclitaxel, andcyclophosphamide.

Other chemotherapeutic agents that may be used include abarelix,aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine,amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live,bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone,camptothecin, capecitabine, carboplatin, carmustine, celecoxib,cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetinalfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel,doxorubicin, dromostanolone, Elliott's B solution, epirubicin, epoetinalfa, estramustine, etoposide, exemestane, filgrastim, floxuridine,fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumabozogamicin, gefitinib, goserelin, hydroxyurea, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferonalfa-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine,meclorethamine, megestrol, melphalan, mercaptopurine, mesna,methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane,mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin,oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase,pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamycin,polifeprosan, porfimer, procarbazine, quinacrine, rasburicase,rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva,temozolomide, teniposide, testolactone, thioguanine, thiotepa,topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracilmustard, valrubicin, vinblastine, vincristine, vinorelbine, andzoledronate.

In certain embodiments, the biological sample comprises FFPEs. Incertain embodiments, the subject's cancer is breast cancer. In certainembodiments, the breast cancer is metastatic. In some embodiments, thebreast cancer is early stage breast cancer. In some embodiments, anycancer that may be sensitive to a HER2 acting agent may be monitored.The HER2 acting agent may be any HER2 acting agent. In certainembodiments, the HER2 acting agent is one of the agents describedherein. For example, in certain embodiments, the HER2 acting agent istrastuzumab. In certain embodiments, the chemotherapeutic agent ispaclitaxel.

Importance of HER2 and p95 in Breast Cancer

Breast cancer is among malignancies with remarkably high risk of brainrelapse. Tsukada, Y. et al., Central nervous system metastasis frombreast carcinoma. Autopsy study. Cancer 52:2349-2354 (1983); Schouten,L. J. et al., Incidence of brain metastases in a cohort of patients withcarcinoma of the breast, colon, kidney, and lung and melanoma. Cancer94:2698-26705 (2002). Brain metastases accompanying breast cancer areassociated with poor prognosis, seriously affect quality of life and arerelatively resistant to systemic therapies. Particularly high risk ofbrain relapse is associated with overexpression or amplification of HER2gene. Hicks, D. G. et al., Breast cancers with brain metastases are morelikely to be estrogen receptor negative, express the basal cytokeratinCK5/6, and overexpress HER2 or EGFR. Am J Surg Pathol. 30:1097-1104(2006); Gabos, Z. et al. Prognostic significance of human epidermalgrowth factor receptor positivity for the development of brainmetastasis after newly diagnosed breast cancer. J Clin Oncol.24:5658-5663 (2006); Gonzalez-Angulo, A. M. et al., Central nervoussystem metastases in patients with high-risk breast carcinoma aftermultimodality treatment. Cancer. 101:1760-1766 (2004).

Currently, the standard component of systemic therapy in HER2-positivebreast cancer patients is trastuzumab, a monoclonal antibody againstextracellular domain of HER2 receptor. Although on average the use oftrastuzumab is associated with considerable progression-free and overallsurvival benefit, only a fraction of HER2-positive metastatic breastcancer patients respond to this agent and a significant proportion ofresponders relapse within one year. Slamon, D. J. et al., Use ofchemotherapy plus a monoclonal antibody against HER2 for metastaticbreast cancer that overexpresses HER2. N Engl J Med. 344:783-792 (2001);Burstein, H. J. et al., Trastuzumab plus vinorelbine or taxanechemotherapy for HER2-overexpressing metastatic breast cancer: thetrastuzumab and vinorelbine or taxane study. Cancer 110:965-972 (2007);Marty, M. et al. Randomized phase II trial of the efficacy and safety oftrastuzumab combined with docetaxel in patients with human epidermalgrowth factor receptor 2-positive metastatic breast cancer administeredas first-line treatment: The M77001 Study Group. J Clin Oncol.23:4265-4274 (2005); Schaller, G. et al., Phase II study of capecitabineplus trastuzumab in human epidermal growth factor receptor2-overexpressing metastatic breast cancer pretreated with anthracyclinesor taxanes. J Clin Oncol. 25:3246-3250 (2007); Robert, N. et al.,Randomized phase III study of trastuzumab, paclitaxel, and carboplatincompared with trastuzumab and paclitaxel in women withHER-2-overexpressing metastatic breast cancer. J Clin Oncol.24:2786-2792 (2006). Increased HER2 total (H2T) levels have been shownto be associated with better response to trastuzumab and prolonged timeto progression in advanced breast cancer patients. Lipton, A. et al.Quantitative HER2 protein levels predict outcome in fluorescence in situhybridization-positive patients with metastatic breast cancer treatedwith trastuzumab. Cancer 116:5168-78 (2010); Toi, M. et al.,Differential survival following trastuzumab treatment based onquantitative HER2 expression and HER2 homodimers in a clinic-basedcohort of patients with metastatic breast cancer. BMC Cancer 10:56(2010).

Importantly, due to a high molecular weight (145,000 Da) and otherphysical and chemical properties, trastuzumab does not cross theblood-brain barrier, and is ineffective in preventing and treating brainmetastases. Pestalozzi, B. C. and Brignoli, S., Trastuzumab in CSF. J.Clin. Oncol. 18:2349-2351 (2000); Stemmler, J. et al., V. Brainmetastases in HER2-overexpressing metastatic breast cancer: comparativeanalysis of trastuzumab levels in serum and cerebrospinal fluid. J ClinOncol. 24:1525 (2006).

However, p95 is a truncated form of HER2 that lacks the trastuzumabbinding site and is therefore thought to confer resistance to tratuzumabtreatment. Sperinde, J. et al., Clin. Cancer. Res. 16(16): 4226-4235(2010). Early data suggested that the presence of p95 correlates to theextent of lymph node involvement, begging the question of causality,whether p95 levels were a result of or contributed to an environmentfavorable to tumor dissemination. See Molina et al., Clin. Can. Res.8:347-353 (2002). Using an indirect measurement of the sub-cellularlocalization of the HER2 intracellular domain (ICD), it has beendemonstrated that intracellular distribution of the HER2 ICD, separatelyshown to correlate with p95 expression, trended (Fisher test p=0.057)toward a correlation with a reduction in RECIST response totrastuzumab-containing treatment. Scaltriti et al., J. Natl Cancer Inst.99:628-638 (2007). Using direct antibody detection of p95, expression ofp95 was found to correlate with reduced progression free survival(HR=1.9; p=0.017) and overall survival (HR=2.2; p=0.012). Sperinde etal., Clin. Cancer. Res. 16(16): 4226-4235 (2010).

Studies have shown that 30-50% of HER2-positive advanced breast cancerpatients will develop brain relapse, with an annual risk of around 10%.See., e.g., Bendell, J. C. et al., Central nervous system metastases inwomen who receive trastuzumab-based therapy for metastatic breastcarcinoma. Cancer 97:2972-2977 (2003); Shmueli, E. et al., Centralnervous system progression among patients with metastatic breast cancerresponding to trastuzumab treatment. Eur J Cancer 40:379-382 (2004);Clayton, A. J. et al., Incidence of cerebral metastases in patientstreated with trastuzumab with trastuzumab for metastatic breast cancer.Br J Cancer 91:639-643 (2004); Lai, R. et al., The risk of centralnervous system metastases after trastuzumab therapy in patients withbreast carcinoma. Cancer 15:810-816 (2004); Lower, E. E. et al.,Increased rate of brain metastases with trastuzumab therapy notassociated with impaired survival. Clin Breast Cancer 4:114-119 (2003);Burstein, H. J. et al., Isolated central nervous system metastases inpatients with HER2 overexpressing advanced breast cancer treated withfirst-line trastuzumab based therapy. Ann Oncol. 16:1772-1777 (2005);Stemmler, H. J., et al., V. Characteristics of patients with brainmetastases receiving trastuzumab for HER2 overexpressing metastaticbreast cancer. Breast 15:219-225 (2006); Gori, S. et al., Centralnervous system metastases in HER-2 positive metastatic breast cancerpatients treated with trastuzumab: incidence, survival, and riskfactors. Oncologist 12:766-773 (2007); Duchnowska, R. et al., Riskfactors for brain relapse in HER2-positive metastatic breast cancerpatients. Breast Cancer Res Treat. 117:297-303 (2009).

Owing to the impaired penetration of trastuzumab through the blood-brainbarrier, brain metastases frequently occur in patients with responsiveor stable disease at metastatic extracranial sites. Nam, B. H. et al.,Breast cancer subtypes and survival in patients with brain metastases.Breast Cancer Res. 10:R20 (2008); Eichler, A. F. et al., Survival inpatients with brain metastases from breast cancer: the importance ofHER-2 status. Cancer 112:2359-2367 2008). On the other hand, owing tobetter control of extracranial metastatic disease, trastuzumab therapywas found to delay the development of brain relapse. Dawood, S. et al.,Defining prognosis for women with breast cancer and CNS metastases byHER2 status. Ann Oncol. 19:1242-1248 (2008); Park, I. H. et al.,Trastuzumab treatment beyond brain progression in HER2-positivemetastatic breast cancer. Ann Oncol. 20:56-62 (2009). Also, continuingtreatment with trastuzumab beyond brain progression results in prolongedsurvival. Church, D. N. et al., Extended survival in women with brainmetastases from HER2 overexpressing breast cancer. Am J Clin Oncol.31:250-254 (2008); Metro, G. et al., Clinical utility of continuingtrastuzumab beyond brain progression in HER-2-positive metastatic breastcancer. Oncologist 12:1467-1469 (2007).

Several retrospective studies have explored clinical and biologicalfeatures associated with a propensity to develop brain relapse inHER2-positive advanced breast cancer patients. Reported adverse factorsincluded the presence of visceral disease, younger age, premenopausalstatus, short disease-free interval after primary therapy and thenegative hormone receptor status. However, the results of particularstudies have been inconsistent and none of these factors or theircombination could allow selecting a subset of HER2-positive advancedbreast cancer patients who might benefit from active surveillance forbrain relapse or from potential preventive strategies. Recently,expression of several genes was found to be associated with increasedrisk of brain relapse both in general population of breast cancerpatients (Bos, P. D. et al., Genes that mediate breast cancer metastasisto the brain. Nature 459:1005-1009 (2009)) and in the HER2-positivesubset (Duchnowska, R. et al., Gene expression analysis for predictionof early brain metastasis in HER2-positive breast cancer patients. JClin Oncol. 26(Suppl.):45s (2008)). However, no robust molecularsignature to predict brain relapse has been developed.

Using the claimed methods described herein, it has been shown for thefirst time that the quantitative assessment of HER2 protein or p95protein in the primary tumor may identify HER2 positive advanced breastcancer patients with particularly high risk of developing brainmetastases during trastuzumab therapy by enabling identification ofsubjects with relatively high levels of HER2 or p95. Using the methodsdescribed herein, it has been determined that relatively high levels ofHER2 or p95 are associated with shorter time to brain metastases (TTBM).Notably, in the multivariate model, quantitative measurement of Her2 andp95, tumor grade, and time to distant progression were the onlypredictors of brain relapse, and only H2T and time to distantprogression were statistically significant predictors of this event inthe most stringently selected subset of HER-2_ patients as assessed byFISH. All other molecular and clinical factors, such as the HER-2/CEP17ratio, HER-2 amplification, hormone receptor status, menopausal status,and age, were not statistically significant correlates of TTBM. Suchdifferential biological effect of H2T levels or p95 levels may be due toinefficacy of trastuzumab in preventing and combating brain metastases.However, it is also possible that better control of extracranial diseasewith this compound may merely provide more time for the clinicalmanifestation of brain relapse (the effect of “unmasking”). The latteris supported by the analysis described herein taking into accountprogressions at other sites as competing events confirmed significantcorrelation between elevated levels of H2T (considered as continuousvariable) and TTBM.

Methods of determining a course of treatment for a high-risk populationof cancer patients are also described herein. For example, a correlationbetween quantitative measurements of HER2 protein expression and therisk of brain metastases in advanced HER2-positive breast cancerpatients administered trastuzumab suggests that H2T assessment mightpotentially select patients for more personalized preventive andtherapeutic strategies in this otherwise high-risk population such as,for example, small molecule drugs, chemotherapy, and/or radiationtherapy. Currently several new compounds with potential prophylactic ortherapeutic activity in brain metastases are being a subject of clinicalinvestigations. In contrast to trastuzumab, small molecule drugs aremore likely to traverse the blood-tumor barrier, although access tobrain metastases may still be impeded. Lockman, P. R. et al.,Heterogeneous blood-tumor barrier permeability determines drug efficacyin experimental brain metastases of breast cancer. Clin Cancer Res.16:5664-5678 (2010). For example, lapatinib has shown promise atprevention in preclinical models and some clinical effectiveness intreating brain metastases (Gril, B. et al., Effect of lapatinib on theoutgrowth of metastatic breast cancer cells to the brain. J Natl CancerInst. 100:1092-1103 (2008); Lin, N. U. et al., Phase II trial oflapatinib for brain metastases in patients with human epidermal growthfactor receptor 2-positive breast cancer. J Clin Oncol. 26:1993-1999(2008)). In a randomized phase III study, the addition of lapatinib tocapecitabine in patients who progressed after trastuzumab therapyresulted in decreased rate of symptomatic brain relapse. Cameron, D. etal., A phase III randomized comparison of lapatinib plus capecitabineversus capecitabine alone in women with advanced breast cancer that hasprogressed on trastuzumab: updated efficacy and biomarker analyses.Breast Cancer Res Treat. 112:533-543 (2008). In addition, pazopanib hasshown activity in brain metastasis prevention in mice injected with HER2cell lines. Gril, B. et al., Pazopanib reveals a role for tumor cellB-Raf in the prevention of HER2+ breast cancer brain metastasis. ClinCancer Res. 17:142-153 (2011).

As small molecule HER2-directed agents gain approval for use earlier inthe progression of breast cancer, quantitative HER2 measurements usingthe methods disclosed herein will be useful in guiding patient care.

EXAMPLES Example 1: Analysis of Subject Samples

Study Group:

This study was approved by the Ethics Committee of the MedicalUniversity in Gdan'sk, the coordinating center. Patients were identifiedthrough computerized hospital systems, protocol enrollment lists, or bymanual search. A study group included a consecutive series ofHER2-positive (IHC 3+, or IHC 2+ and FISH-positive), pathologicallyconfirmed, advanced breast cancer patients from 9 Polish institutions.All patients received at least one dose of trastuzumab with or withoutchemotherapy (typically taxanes, vinorelbine, or capecitabine) betweenDecember 2000 and July 2010. The line of therapy during whichtrastuzumab was first administered was not recorded, although the mediantime from diagnosis of metastatic disease to the initiation oftrastuzumab was 3.4 months (range, 0-49 months). This delay resultedfrom the fact that a substantial proportion of patients receivedtrastuzumab as a second-line or subsequent line of therapy in themetastatic setting. Because the time from diagnosis of metastaticdisease to the initiation of trastuzumab did not correlate with the timeto brain metastasis (TTBM) (p=0.7), this factor was not used forstratification. The majority of patients remained on trastuzumabtreatment until progression, three patients terminated trastuzumabadministration earlier as a result of the occurrence of excessivetoxicity or personal decision, and 20 patients continued trastuzumabtherapy beyond progression.

A total of 164 HER2-positive advanced breast cancer patients wereidentified initially, 22 of whom were subsequently removed from theanalysis due to pre-existing brain metastases (n=6) or because theyreceived trastuzumab in the adjuvant setting (n=16), leaving a studycohort of 142 patients (Table 1).

The following information was extracted from the medical records: dateof diagnosis of breast cancer, previous local and systemic therapy, dateand type of first progression (local, regional, distant), date ofdiagnosis of metastatic disease, dominant site of metastatic disease(soft tissue, bone, viscera), date of diagnosis of brain metastasis,dates on which trastuzumab was received, date of first progression whileon trastuzumab therapy, and date of death or last follow-up visit. Fortumors involving more than one category, the dominant site of distantdisease was classified by the category associated with the worstprognosis, irrespective of the extent of involvement, in the followingorder of increasing gravity: soft tissue, bones, viscera. Because of theretrospective nature of this study, tumor staging was performed usingthe American Joint Committee on Cancer/Union for International CancerControl classification from 1997. The brain metastases includedradiographically confirmed (computed tomography or magnetic resonanceimaging) parenchymal brain lesions. No screening for occult brainlesions was performed; therefore, all metastases were symptomatic ordetected accidentally. Follow-up information was extracted from medicalrecords and tumor registries. Data were collected and stored usingMicrosoft Excel.

The median follow-up time was 68 months (range, 7-144 months) from theinitial diagnosis of breast cancer, 34 months (range, 4-121 months) fromthe first occurrence of metastatic disease and 29 months (range, 1-115months) from the initiation of trastuzumab-containing therapy. Themedian time from the initial diagnosis to first distant relapse was 22months (range 0-103 months) and the median time of trastuzumab therapywas 10 months (range, 1-115 months).

TABLE 1 Subject Characteristics All Subjects No BM BM (N = 142) (N = 93)(N = 49) Characteristic Category N % N % N % Menopausal status post- 7855 52 56 26 53 menopausal pre-menopausal 64 45 41 44 23 47 Dominantmetastatic viscera 89 63 20 22 8 16 site soft tissue 28 20 20 22 4 8bone 24 17 52 56 37 76 unknown 1 1 1 1 0 0 Metastatic Type at diagnosis14 10 9 10 5 10 recurrent 18 18 84 90 44 90 ER positive 55 39 40 43 1531 negative 87 61 53 57 34 69 PgR positive 43 30 29 31 14 29 negative 9567 63 68 32 65 unknown 4 3 1 1 3 6 Grade G3 85 60 49 53 36 73 G1 + G2 5740 44 47 13 27 Pathology Type ductal 127 89 84 90 43 88 lobular 4 3 2 22 4 other 3 2 2 2 1 2 unknown 8 6 5 5 3 6 HER2 protein positive 109 7762 67 47 96 (HERmark H2T)^(a) equivocal 7 5 7 8 0 0 negative 26 18 24 262 4 FISH/CEP17  >2.0 117 82 73 78 44 90 ≤2.0 21 15 18 19 3 6 unknown 4 32 2 2 4 Age at progression median 53 54 50 (yrs) range 25-79 25-79 33-72

HER2+ Classification:

HER2-positive status was determined using semiquantitativeimmunohistochemistry (IHC) at the institutions participating in thestudy. HER2 gene copy number assessment using FISH was performedcentrally at the Department of Biology and Genetics, Medical Universityof Gdansk, Poland. Gene amplification was defined as a FISH ratio(HER2/centromeric probe for chromosome 17 [CEP17] ratio) of more than2.0. The cutoffs used to categorize subjects as HER2 FISH positive, HER2FISH equivocal, and HER2 FISH negative were 1.8 and 2.2, respectively.Expression of estrogen receptor (ER) and progesterone receptor (PR) wasdetermined using IHC, with 10% of nuclear staining considered as apositive result.

VeraTag® Assays:

Quantitative HER2 protein levels (units of relative fluorescence per mm²tumor (RF/mm² tumor)) were measured using the HERmark® assay asdescribed by Lipton et al., Quantitative HER2 protein levels predictoutcome in fluorescence in situ hybridization-positive patients withmetastatic breast cancer treated with trastuzumab. Cancer 116:5168-78(2010) and Larson, J. S. et al., Analytical validation of a highlyquantitative, sensitive, accurate, and reproducible assay (HERmark®) forthe measurement of HER2 total protein and HER2 homodimers in FFPE breastcancer tumor specimens. Patholog Res Int. 2010:814176 (2010). See FIG.1A. The cutoffs used to categorize subjects as HER2 positive, HER2equivocal, and HER2 negative using the HERmark® assay were 10.5 and17.8, respectively. Quantitative p95 protein levels (RF/mm²) weremeasured using the VeraTag assay format illustrated in FIG. 1B, and asdescribed in Sperinde, J. et al., Clin. Cancer. Res. 16(16): 4226-4235(2010).

Tumor Staging:

Due to the retrospective nature of this study, tumor staging wasperformed using AJCC/UICC classification from 1997. Metastatic lesionswere grouped into three categories: soft tissue, bones and viscera. Fortumors involving more than one category, dominant site of distantdisease was classified by the category associated with the worstprognosis, irrespective of the extent of involvement, in the followingorder of increasing gravity: soft tissue, bones, viscera. No screeningfor occult brain lesions was performed; therefore all metastases weresymptomatic or detected accidentally.

Time to Brain Metastasis:

The OS time was calculated from the initiation of trastuzumab-containingtreatment to death (from any cause) or was censored at the end offollow-up. The Kaplan-Meier method was used to estimate the probabilityof brain metastases over time. p-values were calculated for theunivariate analysis using the log-rank test with stratification whereindicated. Cox models were used for multivariate analysis withstratification where indicated. Cox models were also used to estimatethe hazard ratio (HR) and its confidence interval (CI). In themultivariate Cox models and in the univariate Cox model assessing thisparticular variable, time to non-brain progression was used as atime-dependent variable to examine the effect of other types ofprogression on the risk for brain metastases. Analyses controlling forthe competing risks of death (on brain relapse and disease recurrence atall other sites) were performed by the method of subdistribution ofcompeting risks as described by Fine, J. P. and Gray, R. J., Aproportional hazards model for the subdistribution of a competing risk.JASA. 94:496-509 (1999). p-values than 0.05 were considered significant.Statistical analyses were prespecified to the extent possible in astatistical analysis plan and were performed independently by separateteams at Monogram Biosciences, Inc. (South Francisco, Calif.) andInternational Drug Development Institute (IDDI), Inc. (Louvain-la-Neuve,Belgium). Any discrepancies were resolved by agreement among theclinical team in Poland and the statistical teams at Monogram and IDDI.

Brain Metastases and Elevated HER2:

In total, 49 of 142 patients (35%) developed symptomatic brain relapse.Among those 49 patients, the median TTBM was 13 months (95% CI, 9-18months). After start of trastuzumab treatment, brain metastases occurredat the time of first metastatic progression in 20 patients, including 17patients who developed brain relapse during trastuzumab treatment. Theremaining 29 patients developed brain relapse after discontinuation oftrastuzumab treatment. Cumulative 1-year, 2-year and 3-year risk ofdeveloping brain relapse was 19%, 30% and 46%, respectively (95% CI,12%-25%, 22%-39%, and 34%-58%, respectively). The median time fromdisease dissemination to brain relapse was 38 months (range, 1-50months). The median overall survival from the initial diagnosis ofbreast cancer in the overall population was 32 months (range, 1-67months). The median OS time from the initiation of trastuzumab therapyin the overall population was 32 months (95% CI, 28-43 months), withmedian OS of 28 months (95% CI, 16-32 months) and 40 months (95% CI,28-66 months) in the subgroups of patients who did and did not developbrain metastasis, respectively.” Please indicated which sentence shouldbe included.

HER2 amplification was found in 117 of the 138 cases analyzable usingFISH (85%). The quantitative HER2 protein level (H2T) as determined byVeraTag® assay showed 86% concordance (κ=0.55) with HER2/CEP17 ratio(considering negative, equivocal, and positive categories for both H2Tand FISH) (see FIG. 3; n=138). Thus, the two assays classified tumorssimilarly with regards to HER2 status.

Example 2: HER2 Levels Correlate with TTBM

Twelve correlates of TTBM were explored for all subject and thosesubjects determined to be HER2-FISH positive (Tables 2 and 3,respectively), including (a) commonly used clinical variables (age,menopausal status, dominant site of metastatic disease, estrogenreceptor [ER] and progesterone receptor [PgR] levels, tumor grade, HER2status by conventional HER2 FISH measurement), (b) time to non-brainprogression, (c) the novel measurement of continuous HER2 protein level(H2T), considered as a categorical variable using a specific cutoff andalso as a continuous variable, and (d) the novel measurement of p95protein level using a predetermined cutoff.

A higher H2T measurement (assessed either as a continuous variable or asa categorical variable using defined cutoffs) was significantlycorrelated with a shorter TTBM in the entire HER-2 positive patientpopulation (defined as HER-2 positive either by IHC 3+ or by FISHratio>2.0). Two other variables that similarly correlated with TTBM aretumor grade and time from initiation of trastuzumab therapy to non-brainfirst progression. The hazard ratio (HR) between previously defined H2Tpositive and H2T negative groups (see Table 2 notation) was 5.6(p=0.007). However, the so defined H2T-negative group in the study groupwas small (n=26). The best discriminating H2T cutoff value was found tobe 50 RF/mm², with a HR=2.6 (p=0.001). Of note, this value was close tothe median H2T of 58 RF/mm². Continuous H2T was also significantlycorrelated with TTBM (p=0.013), indicating a proportional rise in riskof brain metastases across the entire range of H2T. In contrast to this,neither the cutoff at FISH/CEP17=2.0 nor continuous FISH/CEP17correlated with TTBM (p=0.28 and 0.15, respectively).

As noted above, both H2T and tumor grade were univariate correlates ofTTBM. To confirm that H2T impacted TTBM independent of grade, theanalyses were repeated with tumor grade as a stratification factor.Stratifying for grade, the HR for H2T at a cutoff of 50 RF/mm² was stillsignificant (HR=2.2; p=0.013), although the correlation of continuousH2T with TTBM in the entire population was only trending (p=0.070).

A competing risks analysis was performed to confirm that the occurrenceof death was not impeding the ability to accurately measure thecorrelation of H2T with TTBM. Controlling for death, H2T remained asignificant correlate of TTBM using both the H2T=50 RF/mm² cutoff (HR,2.7; p=0.0009) and a continuous H2T (HR, 2.7; p=0.0066) with HRs similarto or slightly higher than those calculated without controlling fordeath (Table 2) (HRs of 2.6 and 2.3, respectively). H2T assessed eitheras a continuous variable or as a categorical variable using definedcutoffs was not correlated with OS in this patient population. Thisobservation may be due to the small size of the HER2 negative subgroup.

The symbols as set forth in Tables 2 and 3 below refer to the following:

-   -   a. Test for significant difference between any of the three        categories of viscera, bone and soft tissue.    -   b. Time to non-brain progression used as a time-dependent        variable to examine effect of other progressions on risk of        brain metastases.    -   c. Grades 1 and 2 were combined because there was only three        Grade 1 cases.    -   d. VeraTag® HER2 positive status is defined as H2T>17.8, and        VeraTag® HER2 negative is defined as H2T≤10.5, with equivocal in        between these two limits. These cutoffs were previously found to        coincide with central lab determined 95th percentile of        HER2-negatives and 5th percentile of HER2-positives. See Huang        et al., Amer. J. Clin. Pathol. 134:303-311 (2010).    -   e. Insufficient number of events to estimate hazard ratio.        -   There were four cases with unknown PgR, four cases with            unknown FISH and seven cases where FISH spots were too            numerous and clustered to make a reliable count.

TABLE 2 Univariate analysis of time to brain metastases - All SubjectsStratified by Unstratified Grade Variable Category Events/N (%) HRp-value HR p-value Age continuous 49/142 (35%)  0.99 0.6 0.99 0.7Menopausal (MP) post-MP 26/78 (33%) 0.97 0.9 0.94 0.8 status pre-MP23/64 (36%) Dominant metastatic multiple^(a) 49/141 (35%)  — 0.095^(a) —0.088^(a) site ER positive 15/55 (27%) 0.75 0.4 0.86 0.6 negative 34/87(39%) PgR positive 14/43 (33%) 1.1 0.7 1.3 0.4 negative 32/95 (34%)Grade G3 36/85 (42%) 2.4 0.007 — — G1 + G2^(c) 13/57 (23%) HER2 proteinpositive^(d) 47/109 (43%)  5.6 0.007 4.4 0.029 (HERmark ® H2T)negative^(d) 2/26 (8%) FISH/CEP17  >2.0 44/117 (38%)  1.9 0.28 1.4 0.6≤2.0  3/21 (14%) Log (H2T) continuous 49/142 (35%)  2.3 0.013 1.9 0.070Log (FISH/CEP17) continuous 45/131 (34%)  1.7 0.25 1.4 0.5 HER2 proteinH2T > 50 32/65 (49%) 2.6 0.001 2.2 0.013 (H2T) H2T ≤ 50 17/77 (22%) HER2protein H2T > median (44) 33/71 (46%) 2.3 0.005 1.9 0.044 (H2T) H2T ≤median (44) 16/71 (23%) Time to non-brain continuous 49/142 (35%)  2.50.006 2.4 0.010 progression^(b)

TABLE 3 Univariate analysis of time to brain metastases - HER2FISH-Positive Stratified by Unstratified Grade Variable CategoryEvents/N (%) HR p-value HR p-value Age continuous 44/117 (38%)  1.0 0.91.0 1.0 Menopausal (MP) post-MP 23/63 (37%) 0.94 0.8 0.93 0.8 statuspre-MP 21/54 (39%) Dominant metastatic multiple^(a) 44/116 (38%)  —0.10^(a) — 0.12^(a) site ER positive 14/40 (35%) 1.0 0.9 1.1 0.9negative 30/77 (39%) PgR positive 13/35 (37%) 1.4 0.4 1.5 0.22 negative28/79 (35%) Grade G3 33/73 (45%) 2.2 0.022 — — G1 + G2^(c) 11/44 (25%)HER2 protein positive^(d) 44/103 (43%)  —^(e) —^(e) —^(e) —^(e) (HERmarkH2T) negative^(d) 0/11 (0%) FISH/CEP17  >2.0 — — — — — ≤2.0 Log (H2T)continuous 44/117 (38%)  3.0 0.008 2.8 0.022 Log (FISH/CEP17) continuous42/110 (38%)  1.4 0.6 1.2 0.7 HER2 protein H2T > 50 31/63 (49%) 2.60.003 2.3 0.014 (H2T) H2T ≤ 50 13/54 (24%) HER2 protein H2T ≥ median(58) 30/59 (51%) 2.4 0.006 2.1 0.021 (H2T) H2T < median (58) 14/58 (24%)p95 protein p95 > 2.8 30/69 (43%) 2.0 0.037 1.7 0.12 p95 ≤ 2.8 14/48(29%) Time to non-brain continuous 44/117 (38%)  2.4 0.015 2.2 0.025progression^(b)

Example 3: H2T and p95 Levels Correlate with TTBM in a FISH-PositivePopulation

In order to prevent data skewing due to false IHC HER2 positives,parallel analysis was performed in a subset of 117 subjects whose HER2FISH-positive status was centrally determined (Table 3). In this group,H2T (assessed as a continuous variable and using defined cutoffs, withor without stratification by tumor grade) was significantly correlatedwith TTBM, whereas continuous FISH/CEP17 was not (p≥0.6). Within theHER2 FISH-positive population, patients with tumors determined to haveabove-median HER2 protein levels (H2T=58 RF/mm²) were more than 2-foldmore likely to develop brain metastases than those with below medianHER2 protein without (HR, 2.4; p=0.006) and with (HR, 2.1; p=0.021)grade stratification (see FIG. 4A). A slightly larger difference wasseen for the cutoff value of 50 RF/mm² without (HR=2.6; p=0.003) andwith (HR=2.3; p=0.014) grade stratification (see FIG. 4B). In contrast,patients with tumors determined to have HER2 gene amplification above orbelow the median (HER2/CEP17=6.9) had similar likelihood of developingbrain metastases both without (HR=1.3; p=0.4) and with (HR=1.3; p=0.5)grade stratification (see FIG. 4C). Similar results were also observedwith other FISH HER2/CEP17 cutoffs (data not shown).

High levels of p95 expression has previously been shown to correlatewith poor outcomes in trastuzumab-treated breast cancer. Sperinde, J. etal., Clin. Cancer. Res. 16(16): 4226-4235 (2010). Sperinde et al. used ap95 cutoff of 2.8 RF/mm² for a VeraTag® assay format as set forth inFIG. 1B to stratify subjects into high p95 expressing and low p95expression groups. In the current cohort, p95 expression above the p95cutoff of 2.8 RF/mm² was found to correlate with a shorter time to brainmetastasis (Table 3 and FIG. 4D). The 2.8 RF/mm² cutoff was also foundto be the optimal cutoff for differentiating high and low risk of brainmetastasis in this set. Within the HER2 FISH-positive population,patients with tumors determined to have above-cutoff p95 protein levels(p95>2.8 RF/mm²) were about 2-fold more likely to develop brainmetastases than those with below the p95 protein cutoff without (HR,2.0; p=0.037) and with (HR, 1.7; p=0.12) grade stratification (see FIG.4D). This analysis had previously been performed on this same set ofsubject samples before macrodissection to remove non-tumor contaminantscould be accomplished, and the correlation with time to brain metastasiswas found to be consistent. See U.S. Provisional Appl. No. 61/488,028,FIG. 2D.

In order to determine if the H2T cutoff and p95 cutoff are independentvariables, the subset of patients who were determined to have HER2FISH-positive status and H2T expression below the 50 RF/mm² cutoff werefurther assessed based on p95 expression levels. As shown in FIG. 4E,subjects with levels of p95 below the p95 cutoff had a longer TTBM thanthose with p95 levels above the p95 cutoff (HR=2.5, p=0.10). Thus, useof p95 as a variable enabled identification of additional patients atrisk for brain metastasis as compared to H2T measurements alone. Thesmall number of patients assessed likely negatively impacted the pvalue.

A competing risks analysis was performed to confirm that the occurrenceof death was not impeding the ability to accurately measure thecorrelation of H2T with TTBM. Controlling for death, H2T was still foundto be a significant correlate of TTBM for both the H2T=50 RF/mm² cutoff(HR=2.7; p=0.0009) and continuous H2T (HR=2.7; p=0.0066), with HRssimilar to or slightly higher than those calculated without controllingfor death (Table 2: HR=2.6 and HR=2.3, respectively). Additionally, thep95 cutoff was still found to correlate with TTBM while controlling fordeath (HR=1.7, p=0.089).

Example 4: Effect of Tumor Grade on H2T Correlation with Time to BrainMetastases

Because tumor grade was found to be a significant correlate of TTBM onunivariate analysis, and it had some effect on the HR CIs for H2T whenused as a stratification factor, we separately examined the correlationof H2T with TTBM in the subsets of patients with grade 1-2 and grade 3tumors. A Forest plot with hazard ratios for the H2T=50 RF/mm² cutofffor each of the grade subgroups is shown in FIG. 5A. The correlationbetween H2T and TTBM was stronger in the grade 1-2 subset, consistentwith an interaction between H2T and tumor grade (p=0.025). AKaplan-Meier plots for the four subgroups defined by the H2T=50 RF/mm²cutoff and two grade categories is shown in FIG. 5B. TTBM for the foursubgroups defined by the H2T cutoff of 50 RF/mm2 and two gradecategories (combined grades 1-2 versus grade 3) showed similar outcomesfor three of the groups (H2T low-grade 3, H2T high-grade 1-2, H2Thigh-grade 3) as compared to that of the H2T low-grade 1-2 subset, whichhad a significantly lower likelihood of developing brain metastases(log-rank p=0.0012 for comparison of the four subgroups; HR=5.7;p=0.0001 for [H2T>50 RF/mm² or grade 3] vs. [H2T<50 RF/mm² andgrade<3]). A univariate Cox model estimated an HR of 0.17 for the H2Tlow-grade 1-2 patients compared with patients in the three other groups(p=0.0001). Even though the log-rank comparison of the four subgroups(with three degrees of freedom) was also significant (p=0.0012), thisresult should be interpreted cautiously given the multiple comparisonsthat are possible among these four subsets.

To examine whether this was a general phenomenon across the entire rangeof H2T, a similar analysis was performed to examine the correlation ofcontinuous H2T with TTBM within the two subgroups of grades 1+2 or grade3. As shown in FIG. 6A, there was no statistical difference in thecorrelation between continuous H2T and TTBM for the different gradegroups (interaction p-value=0.6). However, as shown in FIG. 6B, a trendtoward a stronger correlation between continuous H2T and TTBM wasobserved in the grade 1-2 patient subgroup than in the grade 3 patientsubgroup in the subset of patients who were confirmed centrally as HER-2positive by FISH (interaction p=0.10).

Example 5: The Impact of Progression Other than Brain Metastases

It has previously been demonstrated that a shorter time to any distantrelapse in HER2-positive advanced breast cancer patients was associatedwith increased risk of developing brain metastases. Similarly, using themethods of the current invention, progression treated as a timedependent variable was found to be significantly associated with TTBM inunivariate analysis (Tables 2 and 3). To further examine the correlationbetween other sites of progression/metastasis and TTBM, multivariatemodels were tested for independent correlation of TTBM with H2T,FISH/CEP17, estrogen receptor (ER), progesterone receptor (PgR), tumorgrade, and progression other than brain metastases as a time-dependentvariable. Multivariate models were fitted with TTBM as the outcome, H2Tand the HER-2/CEP17 ratio as baseline variables, and progression otherthan brain metastasis as a time-dependent variable. The models werestratified by tumor grade and ER and PgR status. H2T and FISH/CEP17 weretested as continuous variables rather than using defined cutoffs toavoid potential overfitting associated with particular cutoffs.

H2T and FISH were further assessed by multivariate analysis and, aashown in Table 4, time to non-brain progression was again found to besignificantly associated with TTBM while controlling for other indicatedvariables. In the subset of patients who were HER2 FISH-positive, acontinuous H2T level (HR=3.3; p=0.024) and time to non-brain progression(HR=2.9; p=0.0056) were found to independently correlate with TTBM,whereas HER2/CEP17 was not. Similar results were found with gradestratification.

TABLE 4 Multivariate analysis of time to brain metastases HER2+ by AllSubjects FISH Variable HR p-value HR p-value Log(H2T) 2.3 0.071 3.30.024 Log(FISH/CEP17) 0.61 0.46 0.45 0.32 Time to non-brain 3.0 0.00352.9 0.0056 progression^(a)

H2T and the other clinical variables set forth in Tables 2 and 3 werealso further assessed by multivariate analysis. H2T and FISH/CEP17 weretested as continuous variables rather than with cutoffs to avoid biasesassociated with any particular cutoff. As shown in Tables 5 and 6, timeto non-brain progression was again found to be significantly associatedwith TTBM while controlling for other factors. In the HER2 FISH-positivesubset, continuous H2T (HR=3.2; p=0.021) and time to non-brainprogression (HR=3.0; p=0.0044) were found to independently correlatewith TTBM. Similar results were found with grade stratification.

TABLE 5 Multivariate analysis of time to brain metastases - All SubjectsStratified Unstratified by Grade Variable HR p-value HR p-value ER 0.600.25 0.62 0.28 PgR 1.7 0.24 1.6 0.28 Grade 1.7 0.14 — — Log(H2T) 2.30.068 2.3 0.071 Log(FISH/CEP17) 0.75 0.67 0.75 0.65 Time to non-brain3.2 0.0019 3.2 0.0019 progression^(a)

TABLE 6 Multivariate analysis of time to brain metastases - HER2FISH-Positive Stratified Unstratified by Grade Variable HR p-value HRp-value ER 0.7 0.44 0.74 0.51 PgR 1.7 0.25 1.6 0.28 Grade 1.7 0.17 — —Log(H2T) 3.2 0.021 3.3 0.021 Log(FISH/CEP17) 0.64 0.56 0.64 0.56 Time tonon-brain 3.0 0.0044 3.0 0.0042 progression^(a)

Example 6: HER2 Protein Level According to Dominant Metastatic Site

Following the finding that H2T correlated with TTBM, further analysiswas conducted to determine if the occurrence of metastases at othersites was also correlated with H2T. Detailed site-specific follow up wasnot available for other metastatic sites, however the dominant site ofmetastasis were known for 141 of the 142 patients. As shown in FIG. 7,the distributions of H2T measurements were not statistically differentbased on the dominant metastatic site (soft tissue, bone andextracranial viscera) (p=0.9). In addition, no correlation was observedbetween the dominant metastatic site and TTBM (p=0.1).

While the preferred embodiments of the invention have been illustratedand described, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

All printed patents and publications referred to in this application arehereby incorporated herein in their entirety by this reference.

That which is claimed is:
 1. A method of treating a subject having aHER2-positive cancer, the method comprising the steps of: selecting asubject who has been determined to have an amount of p95-HER2 protein ina sample of the subject's HER2-positive tumor above a predeterminedcutoff by: (a) providing a p95-HER2 monoclonal antibody produced by ahybridoma cell line having ATCC accession number PTA-9740 (p95.D9.1),PTA-9738 (p95.D3.4), or PTA-9739 (p95.D8.2); (b) providing a bindingcompound that binds to the p95-HER2 monoclonal antibody, wherein thebinding compound comprises a molecular tag covalently attached theretovia a cleavable linkage; (c) quantifying the amount of p95-HER2 proteinin a sample from the subject by the steps of (i) incubating the samplewith the p95-HER2 monoclonal antibody and the binding compound, (ii)treating the sample with a cleaving agent to release the molecular tagfrom the binding compound, and (iii) measuring the amount of releasedmolecular tag as indicative of the amount of p95-HER2 protein in thesample; and (d) comparing the amount of p95-HER2 protein in the sampledetermined in step (c) with the predetermined cutoff, wherein thepredetermined cutoff comprises at least one of (i) a level of p95-HER2protein expression at least two-fold greater than a control cancer cellline having a basal level of p95-HER2 expression, (ii) a level ofp95-HER2 protein expression corresponding to at least a top 30^(th)percentile of p95-HER2 protein expression in a reference cohort ofsubjects having the HER2 positive cancer; and administering aHER2-acting agent and a second form of cancer treatment to the subject,wherein the p95-HER2 protein has a first amino acid corresponding tomethionine 611 of HER2 protein.
 2. The method of claim 1, wherein thepredetermined cutoff comprises a level of p95-HER2 protein expressioncorresponding to at least a top 40^(th) percentile of p95-HER2 proteinexpression in a reference cohort of subjects having the HER2 positivecancer.
 3. The method of claim 1, wherein the control cancer cell linecomprises SKBR3 and/or MCF7.
 4. The method of claim 1, wherein thesecond form of cancer treatment comprises at least one of aHER2-targeted small molecule drug, chemotherapy, or radiation therapy.5. The method of claim 1, wherein the HER2-positive cancer of thesubject has been characterized as HER2-positive based on at least one ofan elevated level of HER2 gene expression, an elevated level of HER2protein level, or HER2 gene amplification.
 6. The method of claim 1,wherein the subject has been characterized as HER2-positive byquantifying the amount of HER2 in the sample using a quantitativeimmunoassay and determining if the amount of HER2 in the sample is abovemedian amount of HER2 determined in the reference population of subjectshaving the HER2-positive cancer.
 7. The method of claim 1, wherein theHER2-positive cancer of the subject comprises breast cancer.
 8. Themethod of claim 7, wherein the HER2-positive cancer of the subjectcomprises primary breast cancer.
 9. The method of claim 1, wherein theHER2-acting agent is a monoclonal antibody.
 10. The method of claim 9,wherein the monoclonal antibody is trastuzumab.
 11. The method of claim1, wherein the subject has increased risk of metastasis as compared to acontrol subject that has a p95-HER2 protein amount below thepredetermined cutoff.